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.     

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.     

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.     

Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy-1.

To examine the extent to which protein structure and tissue-type influence glycosylation, we have determined the oligosaccharide structures at each of the three glycosylation sites (Asn-23, 74 and 98) of the cell surface glycoprotein Thy-1 isolated from rat brain and thymus. The results show that there is tissue-specificity of glycosylation and that superimposed on this is a significant degree of site-specificity. On the basis of the site distribution of oligosaccharides, we find that no Thy-1 molecules are in common between the two tissues despite the amino acid sequences being identical. We suggest, therefore, that by controlling N-glycosylation a tissue creates an unique set of glycoforms (same polypeptide but with oligosaccharides that differ either in sequence or disposition). The structures at each of the three sites were also determined for the thymocyte Thy-1 that binds to lentil lectin (Thy-1 L+) and for that which does not (Thy-1 L-). Segregation of intact thymus Thy-1 into two distinct sets of glycoforms by lentil lectin was found to be due to the structures at site 74. Analysis of oligosaccharide structures at the 'passenger' sites (23 and 98) suggests that either Thy-1 L+ and Thy-1 L- molecules are made in different cell-types or that the biosynthesis of oligosaccharides at one site is influenced by the glycosylation at other sites.     

Purification of the Thy-1 molecule from rat brain.

The present paper describes the characterization of the Thy-1 molecule from rat brain. The molecule was recognized by its antigens, which could be solubilized from brain membrane with deoxycholate. In the solubilized form the Thy-1 antigens were associated with a homogenous component with the following hydrodynamic properties: s20,w=2.2s,v=0.72ml/g and Stokes radius=3.0nm. The mol.wt. of the deoxycholateantigen complex was estimated to be 27000; these values are not significantly different from those obtained thymocyte Thy-1. Brain Thy-1 was further purified by affinity chromatography with lentil lectin coupled to Sepharose 4B, and more than 80% of the antigen was bound. The material eluted with methyl alpha-D-glucopyranoside was then filtered on a column of Sephadex G-200, and only one glycoprotein was found in the antigenically active fraction. On sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the glycoprotein was very similar to the Thy-1 from thymocytes that binds to lentil lectin. Its apparent mol.wt. on 12.5% acrylamide gels was 24000, and it electrophoresed as a symmetrical band. Brain Thy-1 was antigenically indistinguishable from thymocyte Thy-1 when analysed with rabbit antisera raised against brain or thymocyte Thy-1.     

Spectrum of reactivity of monoclonal antibodies against specific T-suppressors

Monoclonal antibodies (MoAb) against specific T-suppressors CI and C4 are characterized by their reactivity with normal lymphoid cells and some tumour cell lines cultivated in vitro. MoAb CI and C4 react with T and B cells from spleen and lymph nodes. The amount of CI and C4 T and B subsets are equal in the spleen (25-29%), while lymph node T-lymphocytes contain twice as much CI and C4 cells than B-lymphocytes (40 and 20%, respectively). In the thymus CI is expressed mostly on immature (cortical) thymocytes and C4--on the mature (medullary) thymocytes. CI is expressed on some T-lymphoma cell lines (BW 5147, EL4, LBRM33), but not on thymoma RDM4 and mastocytoma P815. C4 is not found on the above cell lines but is expressed on the intermediate filament of mouse and quail fibroblasts and in lymphoid cells. This cross-reactivity may result from the existance of similar determinants in cytoskeleton proteins and lymphocyte membranes or from the intermediate filament expression on T-suppressor cellular membranes, but not on other functional T subsets.     

Expression of cell surface antigens by suppressor T cell hybridomas. I. Comparison of phenotype and function

The phenotypic expression of cell surface markers by T cell hybridomas that elaborate suppressor factors specific for the polymers L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) or L-glutamic acid50-L-tyrosine50 (GT) has been analyzed. We found that determinants encoded by the I-J subregion of the H-2 complex were borne on the surface of these hybrid cells and on the factors they secrete, whereas I-J determinants were not expressed by the AKR thymoma fusion parent, BW5147. The level of expression of I-J determinants fluctuated widely depending upon culture conditions, but I-J products and other cell surface markers of normal T cells could be quantitatively increased, or induced to appear, by treatment of the hybridomas with chemical agents, such as dimethyl sulfoxide (DMSO) or phorbol myristate acetate (PMA). In contrast, the surface expression of the viral product gp70 was decreased by the same treatment. Using chemical induction, we typed BW5147, a group of antigen-specific suppressor T cell hybridomas, and two control hybridomas for expression of I-J, Thy-1, Lyt, and H-2K alloantigens. Also, a haplotype-specific hybridoma that produces an antigen-nonspecific factor was analyzed. The results demonstrated that BW5147 failed to express I-J or Lyt alloantigens but expressed Thy-1.1 and H-2Kk gene products. The pattern of expression of these antigens by T cell hybridomas was very complex, but three conclusions could be drawn: 1) Good correlation exists between the expression of certain I-J determinants and the ability of T cell hybridomas to produce suppressor factor. 2) The expression of Thy-1, Lyt, or H-2Kk determinants is variable, and no correlation was found between expression of these antigens and the ability to produce active suppressor factors. 3) I-Jk products contributed by the AKR thymoma fusion partner are expressed by T cell hybridomas.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. II. Establishment of a T cell hybrid clone constitutively producing killer-helper factor(s) (KHF) and functional analysis of released KHF

T cell hybridoma lines were constructed by fusion of Mycobacterium tuberculosis-primed and boosted BALB/c T cells with the AKR-derived T lymphoma cell line BW5147. Certain of the hybridomas prepared in this manner secreted constitutively into their culture supernatants biologically active molecules that displayed precursors of cytotoxic T cell activating properties characteristic of killer-helper factor (KHF). Cell surface analysis revealed that the hybridomas were indeed somatic cell hybrids between the two respective partner cells used for fusion. KHF properties of these hybridoma supernatants were verified by their capacity to stimulate peanut agglutinin-binding (PNA+) C3H/He thymocytes to respond in vitro to 2,4,6-trinitrophenyl(TNP)-modified syngeneic stimulator cells in conjunction with suboptimal doses (10 U/ml) of interleukin 2 (IL 2) for the generation of H-2-restricted, TNP-reactive cytotoxic T cells. The biologically active molecules secreted by a T cell hybrid clone (2Y4) were, like conventional KHF, distinct from IL 1, IL 2, or immune interferon (IFN-gamma). The partially purified KHF derived from 2Y4 cells shows activity at apparent m.w. range of 34,000 to 60,000 on gel permeation, and is relatively homogeneous with respect to isoelectric point, which was approximately 4.5 to 4.7. The partially purified 2Y4-KHF is able to augment proliferation of as well as the expression of IL 2 receptors on PNA+ thymocytes in conjunction with IL 2. Finally, addition of 2Y4-KHF on day 0, followed by the addition of IL 2 on day 2 for 7 days of culture was effective in generating potent CTL responses, whereas addition of IL 2 on day 0, followed by the addition of 2Y4-KHF on day 2 to the culture was ineffective.     

Isolation of monoclonal antibodies to antigen Lyt-3.2

A hybridoma producing monoclonal antibodies (McAb) NATF9.9 (F9) was obtained from fusion of murine myeloma X63 and splenocytes of AKR mice immunized with a single intravenous injection of 5 X 10(7) thymocytes of CBA mice. F9 McAb were cytotoxic for 80% thymocytes, 10% splenocytes, 20% lymph node cells, 85% cortical and 32% medullary thymocytes of CBA, C57BL/6, BALB/c, DBA/2 and SJL but not for the cells of C58 and AKR mice. F9 McAb reacted only with T cells and did not react with B cells and EL4 thymoma cells (Thy-1.2+, Lyt-1+2-3-). The proportion of F9+ cells accounts for about 40% among T lymphocytes of the lymph nodes and spleen as tested by flow-type cytometry. Lymph node cells treated with F9 McAb plus complement completely lost their reactivity with rat anti-Lyt-2 McAb and only partly (by 30%) with anti-Lyt-1 McAb. The reactivity pattern of F9 McAb attests to their specificity for Lyt-3.2 antigen.     

Presence of an antigen-specific T cell subset that forms IgE-suppressive factor and IgG-suppressive factor on antigenic stimulation

B6D2F1 mice were given three i.v. injections of ovalbumin (OA), and antigen-specific T cell clones were established from their spleen cells. One of the FcR+ T cell clones formed IgE-binding factors on incubation with OA-pulsed syngeneic macrophages. Neither soluble antigen nor macrophages alone induced factor formation. T cell hybridomas were constructed by fusion of the antigen-specific T cell clone with BW 5147 cells. Among 11 T cell hybridomas established, six clones produced IgE-binding factors on incubation with OA-pulsed BDF1 macrophages. Mouse IgE also induced the same hybridoma to form IgE-binding factors. The majority of IgE-binding factors formed by two T hybridomas and by those produced by the parent T cell clone had affinity for peanut agglutinin but for neither lentil lectin nor Con A. These hybridomas and the original T cell clone spontaneously released glycosylation-inhibiting factor, which inhibits the assembly of N-linked oligosaccharide(s) on IgE-binding factors. On antigenic stimulation, the T cell hybridomas produced both IgE-binding factors and IgG-binding factors. The IgE-binding factors consisted of three species with m.w. of 60,000, 30,000, and 15,000. Both the 60K and 15K IgE-binding factors selectively suppressed the IgE response of DNP-OA-primed rat mesenteric lymph node cells, whereas IgG-binding factors selectively suppressed the IgG response. The results indicate that antigen-primed FcR+ T cells produced IgE-suppressive factors and IgG-suppressive factors on antigenic stimulation. However, the T cell hybridomas were not committed to suppressive activity. When the hybridomas were stimulated by antigen in the presence of glycosylation-enhancing factor (GEF), the 60K, 30K, and 15K IgE-binding factors formed by the cells selectively potentiated the IgE response. IgG-binding factors formed by the cells in the presence of GEF failed to suppress the IgG response. It appears that antigen-specific FcR+ T cells regulate the antibody response through the formation of Ig-binding factors, but that the function of the cells could be switched from suppression to enhancement, depending on the environment of the cells.     

Cytofluorometric analysis of R-Thy-1. antigens in various rat lymphocytes with different electrophoretic mobility and organ distribution.

Small bone marrow lymphocytes, which had been previously enriched by velocity sedimentation, thymocytes, lymph node cells and spleen cells were electrophoretically separated, stained with fluorescein conjugated rabbit a-rat-Thy-1. globulin and their fluorescence intensities analyzed with a flow cytophotometer. Thy-1. antigens were found in 80% of the bone marrow small lymphocytes showing low electrophoretic mobility (EPM), in all thymocytes, about 80% of which show low and the rest medium to high EPM, and in a few lymph node cells of high EPM. Thy-1. positive cells were not observed in the spleen. All fluorescence intensity histograms obtained were modal and could be properly fitted with normal curves showing coefficients of variation (C.V.) in the range of 20% to 30%. It was observed that the thymocytes of low EPM had an antibody binding affinity significantly different from that of the other stained lymphocytes. Moreover the surface antigen density decreased in the sequence: thymocytes of low EPM, bone marrow lymphocytes of low EPM and thymocytes of high EPM. The fluorescence intensity of stained lymph node cells of high EPM appeared similar to that of thymocytes of high EPM but was not evaluated precisely. Thus the two dimensional cell analysis provided by a combination of EPM and surface fluorescence of Thy-1.+ cells, allows the characterization of different lymphocyte populations which cannot be clearly identified with normal one dimensional techniques. The biological significance of the results is discussed briefly.     

Analysis of H-2-linked immune responses involved in resistance to AKR tumor growth

H-2-associated immune response gene(s) govern resistance to growth of a spontaneous AKR lymphoma, BW5147. The antigenic specificities recognized by the anti-BW5147 humoral response have been characterized and include: Thy-1, a T -cell differentiation antigen; gp70, a retroviral envelope protein; and several previously uncharacterized proteins, including a 78 000 molecular mass protein, p78, which is restricted to expression on BW5147 cells and five phosphoproteins with molecular masses of 33 000, 29 000, 23 000, 17 000, and 16 000. Only mice which are able to respond to Thy-1, p78, and the phosphoproteins can survive an inoculation of BW5147. Thus, resistance to BW5147 is complex and involves multiple antigens with possible roles in tumor rejection.Abbreviations used in this paper DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - Ir immune response - MuLV murine leukemia virus - NMS normal mouse serum - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

The Pro-451 to Leu polymorphism within the C-terminal tail of P2X7 receptor impairs cell death but not phospholipase D activation in murine thymocytes

The P2X family of ATP receptors (P2XR) are ligandgated channels that have been proposed to regulate cell death of immature thymocytes. However, the nature of the P2XR subtype involved has been controversial until recently. In agreement with previous studies, we found that extracellular ATP (ATPe) induces a caspase-dependent apoptosis of BALB/c thymocytes, as observed by DNA fragmentation. Additionally, ATPe induces a predominant caspase-independent thymocytes lysis characterized by plasma membrane disruption. Both responses to ATPe can be induced by a potent P2X7R agonist, benzoylbenzoyl-ATP, whereas P2X7R antagonists, oxidized ATP and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, inhibited the effect of ATPe. These results are further supported by observations where disruption of the P2X7R gene (P2X7R(-/-) mice) completely abolishes thymocytes death induced by ATPe. Interestingly, the natural P451L mutation in the C-terminal tail of P2X7R present in C57BL/6 mice, which impairs ATPe-dependent pore formation in T lymphocytes, significantly reduces thymocytes death triggered by ATPe. Furthermore, we found that P2X7R from BW5147 thymoma cells also harbors this point mutation, accounting for their insensitivity to ATPe-induced cell death. Concentrations of ATPe effective in inducing cell death also increase phosphatidylcholine-hydrolyzing phospholipase D (PC-PLD) activity in BALB/c thymocytes through the stimulation of P2X7R. However, in contrast to ATPe-induced cell death, PC-PLD activation is totally Ca(2+)-dependent. Moreover, the stimulation of PC-PLD by ATPe is not affected by the P451L mutation present in C57BL/6 thymocytes and BW5147 cells, suggesting that cell death and PC-PLD activity are regulated through distinct domains of the P2X7R. Finally, the inhibition of ATPe-induced PC-PLD stimulation does not affect thymocytes death. Altogether, these data suggest that P2X7R-induced thymocytes death is independent of the stimulation of PC-PLD activity.     

Mouse IgA Fc receptor on CD3+ T cells. Molecular forms of IgA that bind to a 38-kDa Fc alpha R protein and development of an anti-Fc alpha R antisera

Previous studies have shown that splenic T cells from mice that bear IgA myelomas, as well as certain T cell lines, express receptors for the Fc of IgA, and are termed Fc alpha R. In this study, we have isolated and characterized two CD3+ T cell lines derived by fusion of murine Peyer's patch (PP) CD4+ T cells with the BW 5147 lymphoma cell line. These cell lines, designated PPT4-6 and PPT4-16, were shown to bind monomeric or dimeric IgA, whereas the fusion partner did not bind either form of IgA. However, polymeric IgA (m.w. 600,000) bound equally well to all three cell lines. Similar results were also obtained with two known Fc alpha R+ T cell lines, ThHA1 nos. 9 and 10. Immunoprecipitation studies with IgA on PPT4-16 and ThHA1 no. 9 have shown that IgA binds to a 38-kDa protein. A rabbit antiserum was prepared to a 38-kDa fraction of Fc alpha R+ T cell membranes, and heterophilic antibody was removed from the antiserum by adsorption with mouse thymocytes, BW 5147 and R1.1 lymphoma. The antiserum bound to both PPT4-16 and ThHA1 no. 9 as well as to other Fc alpha R+ T cells, but did not bind to thymocytes or to the T lymphomas R1.1 or BW 5147. The antiserum appeared specific for the Fc alpha R, because it failed to block binding of anti-CD3 (145 2C11) or other surface molecule-specific antibodies. Further, competitive inhibition studies with IgA and anti-Fc alpha R (38 kDa) showed that preincubation of Fc alpha R+ T cells with the anti-38-kDa protein completely eliminated IgA binding, whereas IgA partially blocked the binding of the anti-Fc alpha R antibodies to the cell membrane. Immunoisolation with the anti-Fc alpha R antibody of radioiodinated cell membrane proteins from Fc alpha R+ T cells, but not from Fc alpha R- cells, gave a distinct band at 38 kDa. To further test the specificity of this antiserum, we have isolated T cells from spleens of IgA-myeloma bearing mice, and tested the phenotype and IgA binding. A subset consisting of 15 to 20% of CD3+, CD8+ T cells was found that bound monomeric or dimeric IgA. Further, the anti-Fc alpha R antiserum also recognized this CD8+ T cell subset, and preincubation of the cells with antibody resulted in their failure to bind IgA. Our results indicate that the Fc alpha R on T cell lines derived from PP is a 38-kDa protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel ganglioside expressed by mouse hematopoietic cell lines.

Mouse progenitor T cell-derived cell lines were established by fusion of cells of hematopoietic organs such as bone marrow and fetal liver with T lymphoma (BW5147) to determine their characteristic cell-surface components. The hybridomas with the phenotype of Thy-1+, CD3-, CD4-, CD8- and expression of T cell receptor gene mRNA (BM216 and FL339) were selected for progenitor T cell-derived cell lines, and their ganglioside compositions were studied. A ganglioside component with a mobility slightly faster than that of bovine brain GD1a on high-performance thin-layer chromatography was found in the cell extracts of these cell lines as one of the most abundant components and was absent in the extract of the parental cell line (BW5147). The structure of the ganglioside was determined to be: NeuAc alpha-Gal beta-Gal beta-Gal alpha-Gal beta-Glc beta-ceramide. Gangliosides with such a sequence have never been found before, suggesting the possibility that the ganglioside is expressed as a surface marker of the cells in hematopoietic organs committed to a specific cell lineage, presumably to T cell lineage. cells in hematopoietic organs committed to a specific     

Expression of Thy-1 and effect of phosphatidylinositol-specific phospholipase C on primate and murine cell lines

BALC/c mice were immunized with isolated human brain Thy-1. The antisera at an appropriate dilution only reacted with a doublet of an apparent molecular weight (MW) around 25,000 among all the glycoproteins of brain tissue isolated by lentil lectin affinity chromatography when tested by enzyme-linked immunosorbent assay and immunoblotting. When the antisera were used to test a number of human cell lines and a marmoset T-cell line (70N2) by flow cytometry, an astrocytoma cell line (U-373), a T-lymphoblastoid cell line (MOLT-3), and two cutaneous T-lymphoma cell lines (HUT-78 and HUT-102) as well as the 70N2 cells were stained. However, a B-lymphoma cell line (Raji), a plasmacytoma cell line (HMy2), and normal peripheral blood lymphocytes were negative. When the positive cells were treated with phosphatidylinositol-specific phospholipase C, a significant decrease in both stain intensity and percentage of positive cells was demonstrated by immunofluorescence. Although Thy-1 expression in human lymphoid system is currently thought to be confined in early T- and B-lymphocyte development, our data suggest that well-differentiated T cells with mature phenotypes such as HUT-78 and HUT-102 which may be considered as tumor counterparts are also capable of expressing Thy-1, presumably after certain stimulation.     

Regulatory effects of human IgE-binding factors on the IgE response of rat lymphocytes

Normal human peripheral blood T cells were propagated in the presence of human interleukin 2, and activated cells were incubated with human IgE-dimer to induce IgE binding factor formation. The cells were then fused with a mutant of the human T cell line CEM. Five of the T cell hybridomas formed IgE binding factors upon incubation with human IgE-dimer. Because IgE binding factors formed by the human T cell hybridomas had affinity not only for human IgE but also for rat IgE, the biologic activities of the factors were evaluated by using antigen-primed rat mesenteric lymph node (MLN) cells. When parent T cells were propagated with crude IL 2, which contained glycosylation enhancing factor (GEF), IgE binding factors formed by all of the five hybridomas had affinity for Con A, but only a fraction of the factors bound to lentil lectin. The 60,000 and 15,000 IgE binding factors formed by two representative hybridomas, i.e., 166A2 and 166G11, selectively potentiated the IgE-forming cell response of rat MLN cells. When parent T cells were obtained by propagation with purified IL 2, which did not contain GEF, and the cells were incubated with IgE-dimer in the presence of glycosylation inhibiting factor (GIF), T cell hybridomas constructed from the cells formed IgE binding factors that lacked affinity for Con A but bound to peanut agglutinin (PNA). The 30,000 IgE binding factors formed by two of such hybridomas, 398A3 and 400G2, selectively suppressed the IgE response of rat MLN cells. It was also found that the biologic activities and carbohydrate moieties of human IgE binding factors could be switched by changing the culture conditions of the hybridomas. When the 166A2 hybridoma was cultured with human IgE in the presence of bradykinin, essentially all of the IgE binding factors that were formed by the cells bound to lentil lectin, and the factors that were formed in the presence of bradykinin exerted higher potentiating activity than those obtained in the absence of bradykinin. On the other hand, IgE binding factors formed by the same cells in the presence of GIF had affinity for PNA, and selectively suppressed the IgE response of rat MLN cells.     

Phenotypic characterization of thymic prelymphoma cells of B10 mice treated with split-dose irradiation

Using an intrathymic injection assay on B10 Thy-1 congenic mice, it was demonstrated that thymic prelymphoma cells first developed within the thymuses from 4 to 8 days after split-dose irradiation and were detected in more than 63% of the test donor thymuses when examined at 21 and 31 days after irradiation. Moreover, some mice (25%) at 2 mo after split-dose irradiation had already developed thymic lymphomas in their thymuses. To characterize these thymic prelymphoma cells, the thymocytes from B10 Thy-1.1 mice 1 mo after irradiation were stained with anti-CD4 and anti-CD8 mAb and were sorted into four subpopulations. These fractionated cells were injected into the recipient thymuses to examine which subpopulation contained thymic prelymphoma cells. The results indicated that thymic prelymphoma cells existed mainly in CD4- CD8- and CD4- CD8+ thymocyte subpopulations and also in CD4+ CD8+ subpopulation. T cell lymphomas derived from CD4- CD8- prelymphoma cells had mainly CD4- CD8- or CD4- CD8+ phenotypes. T cell lymphomas developed from CD4- CD8+ prelymphoma cells mainly expressed CD4- CD8+ or CD4+ CD8+ phenotype. T cell lymphomas originating from CD4+ CD8+ prelymphoma cells were mainly CD4+ CD8+ but some CD4- CD8+ or CD4+ CD8- cells were also present. These thymic prelymphoma cells were further characterized phenotypically in relation to their expression of the marker defined by the mAb against J11d marker and TL-2 (thymus-leukemia) Ag, which is not expressed on normal thymocytes of B10.Thy-1.2 or B10.Thy-1.1 strain, but appears on the thymocytes of lymphomagenic irradiated mice. The results indicated that the prelymphoma cells existed in J11d+, TL-2+ cells.     

Hydrophilic anchor-deficient Thy-1 is secreted by a class E mutant T lymphoma

To investigate the mechanism of glycophospholipid anchoring of the surface antigen Thy-1, we have undertaken a comparative biosynthetic study using a wild-type Thy-1+ murine T lymphoma (BW5147) and a mutant T lymphoma (class E) that synthesizes Thy-1 but fails to express it on the plasma membrane. Labelling experiments with D-[2-3H]mannose demonstrate that, unlike the wild type, the mutant cells are secreting large amounts of Thy-1 and that the secreted molecules are hydrophilic. Moreover, unlike the wild type, they fail to incorporate [3H]palmitic acid into Thy-1. Both wild-type and mutant cells do incorporate labeled galactose and fucose into Thy-1. We conclude that the lack of surface expression of Thy-1 by this mutant results from the failure to add anchor components to Thy-1.