Hybridization between T and B lymphoma cell lines.

The AKR thymoma line BW 5147 has been successfully hybridized with the IgM-bearing (BALB/c x NZB)F1 B lymphoma line WEHI 231. In the hybrids formed, T-cell characteristics were dominant, i.e. there was no expression of IgM but continued expression of Thy-1.1 in eight out of eight lines. Moreover, in two out of eight lines, the Thy-1.2 allele was also expressed. We conclude that, in its ability to hybridize, BW 5147 is not restricted to cells of similar ontogenetic origin (i.e. T cells) and that fusion with the thymoma can lead to suppression of B-cell gene expression and derepression of genes for T-cell markers.     

In vitro establishment and some properties of AKR thymoma cell lines.

Lymphoid cell lines could easily be directly established in vitro from spontaneous thymomas of aged AKR mice. The established lines all carried the Thy-1.1 antigenic marker of T lymphocytes. They differed markedly, however, from one another in several respects. Thus, they showed high or low quantity of Thy-1.1 antigens per cell, high or low sensitivity to cortisone and thymidine, as well as differing morphology and growth rate. The observed heterogeneity suggests that such AKR thymoma cell lines may provide a valuable tool to study T lymphocyte properties and the biology of the AKR thymoma, in particular non-immune and immune mechanisms influencing thymoma cell growth.     

Immunological activity of a T hybrid line. I. Production of an H-2-related suppressor factor with specificity for sheep red blood cells.

T lymphocyte hybrid lines have been produced by fusion of the thymoma BW 5147 with spleen cells of C57BL/10 mice primed to sheep red blood cells (SRBC). The supernatant (culture fluid) of a T hybrid designated A 1 was able to suppress the primary (IgM) and secondary (IgM and IgG) antibody responses to SRBC in vitro. The suppressive activity of supernatants could be titrated to 50% end points at final dilutions of up to 1 : 270. The suppression affected only SRBC and haptens coupled to SRBC, except when used at high concentration when some nonspecific suppression was observed. Absorption of the A 1 supernatant with SRBC removed all activity, while several other species of red cells failed to do so. The suppressor factor present in A 1 supernatant was not removed by anti-Ig adsorbents, but was removed by anti-H-2 antibodies reacting specifically with the haplotype of the spleen cells used in the fusion (H-2b). The cellular target of action of the factor was apparently a B cell, based on absorption with different cell populations. No genetic restrictions in the activity of the factor were found. A 1 cells carried H-2 and Thy-1 alleles of both parental cells and formed rosettes with SRBC.     

T lymphocyte tissue culture lines produced by cell hybridization.

This study describes the generation of permanent T cell tissue culture lines by cell fusion techniques. The AKR strain-derived T cell tumor BW 5147 was hybridized in the presence of polyethylene glycol with various T cell populations isolated from antigen-sensitized mice. Surface analysis of resulting hybrid cell lines showed expression of both Thy-1.2 and H-2 antigens which are characteristic for the lymphocyte used for fusion. In contrast, in hybrids derived from spleen cells neither expression of Ig nor of B cell-typicyl Ia determinants was found suggesting either preferential hybridization of BW 5147 cells with T lymphocytes or extinction of B cell markers in hybrid cells. These hybrid lines which may display the immunological properties of the T cell population chosen are presently investigated for their antigen reactivity.     

Production and characterization of cytotoxic Thy-1 antibody-secreting hybrid cell lines. Detection of T cell subsets.

Responses to Thy-1 were used as a model system to examine parameters which affect the production of antibody-secreting lines derived from somatic cell hybridization. Experiments with the Thy-1.1 response revealed that the frequency of clones producing Thy-1.1 antibodies is a constant of 4 to 6% for each 10000 plaque-forming cells (PFC) input in the fusion cell mixture, regardless of the maturational stage of the response. Therefore, PFC responses to Thy-1 were optimized by studying variables in the choice and dose of antigen, the response kinetics and in the fusion procedures. Thus, to produce Thy-1.1 antibody-secreting cell lines, we used (a) spleen cells at the peak of the PFC response, (b) xenogeneic (rat) rather than allogeneic donors, (c) secondary rather than primary responses and (d) high ratios of NS-1 to spleen cells. For the reproducible production of Thy-1.2 antibody-secreting hybridomas, PFC responses to Thy-1.2 were similarly optimized in AKR mice. Response kinetics and antigen dose were shown to be very critical parameters. By varying the number of cells used for priming, it was revealed that doses only slightly higher than optimal produced a dramatic hyporesponsiveness in the subsequent secondary response. Using the above information, hybrid lines secreting antibody to Thy-1.2 were obtained reproducibly and one line, F7D 5, which secretes a cytotoxic IgM antibody was characterized in detail since a monoclonal antibody may differ from conventional antisera for immunochemical and genetic reasons. Serologically, F7D 5 Thy-1.2 antibody was found to behave as a conventional Thy 1.2 alloantiserum, At high dilutions however, the antibody can be used to discriminate long-lived T cells (adult thymectomized mice) from newly produced T cells (antilymphocyte antiserum-treated mice). Functionally, in numerous T cell-dependent assays both in vivo and in vitro, including helper, suppressor and cytotoxic T cell functions as well as responses to mitogens and antigens, the F7 D 5 antibody behaved as a potent and absolute T cell-depleting agent. This cell line and some anti-Thy-l.1 producing lines are available for research purposes.     

Idiotypic and fine specificity analysis of a (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific suppressor T cell hybridoma at the level of cell surface structures, isolated receptor material and functional suppressor factor

The (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific T suppressor cell hybridoma 7C3-13 was established by fusing splenic B10.BR T cells enriched on NP-coated petri dishes with the AKR thymoma BW5147. 7C3-13 was selected by anti-NPb idiotypic and anti-I-Jk antibodies in microcytotoxicity tests. The hybridoma expressed H-2k, I-Jk, Qa-1, Thy-1.1 as well as idiotypic (binding site-related) and framework Ig VH determinants, while it was negative for I-A, I-E/C, Thy-1.2, Lyt-1, Lyt-2 and Ig constant region determinants. Hapten-binding receptor material could be isolated from 7C3-13 cells on NP-coupled nylon nets and functionally active T suppressor factor (TsF) could be extracted from the hybridoma. Both types of soluble molecules express NPb idiotype, but the TsF carries I-J determinants in addition while the isolated receptors do not. The molecular weight of the isolated receptor material is 80 000, that of the TsF activity is 27 000 and 57 000-64 000, respectively. We thus were able to show that NP-binding molecules can be obtained in the form of cellular surface receptors, isolated receptor material and extracted TsF from one and the same, monoclonal, cell source.     

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.     

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.     

Production and characterization of cloned T-cell hybridomas that are responsive to Rickettsia conorii antigens.

T-cell hybridomas produced by the fusion of Rickettsia conorii immune T cells to the AKR thymoma BW 5147 produced interleukin-2 when stimulated with the antigens of three different R. conorii strains. One cloned hybridoma responded only to R. conorii antigens, whereas a second and third cloned hybridoma also responded to the antigens of Rickettsia rickettsii Sheila Smith and Rickettsia sibirica 246, respectively. Antigen responses required antigen-presenting cells, and this interaction was major histocompatibility complex restricted. Fluorescence-activated cell-sorter analysis demonstrated that all three hybridomas were of the Thy-1.2+, Lyt-2- phenotype and that two of the three were L3T4+. These data demonstrated the presence of an antigenic epitope that is R. conorii species specific and other epitopes that are common to various members of the spotted fever group which can stimulate interleukin-2 production by T-cell hybridomas.     

Antigen density on target cells determines the immunosuppressive potential of rat IgG2b monoclonal antibodies

The current studies were designed to determine the relevance of T cell antigen density, besides antibody isotype, with regard to the success of antibody serotherapy. We compared the immunosuppressive effects of two rat IgG2b monoclonal anti-Thy-1 antibodies, RmT1 and 30-H12, with distinct binding sites in a graft-vs.-host disease (GVHD) model of fully H-2 and I-A region-mismatched bone marrow transplantation, making use of the difference in Thy-1.2 antigen density between homozygous (BALB/c) and heterozygous (BALB/c X AKR/J)F1 GVHD-promoting donor cells. Antibodies RmT1 (directed against a monomorphic determinant on mouse Thy-1) and 30-H12 (reactive with the Thy-1.2 allele-specific determinant) did not differ in their anti-GVHD activity with regard to Thy-1.2 homozygous grafts. However, in the region of a critical number of binding sites a small difference in the amounts of the two antibodies bound (about 8 X 10(3) IgG molecules/cell) obviously accounts for a great difference in anti-GVHD activity. This is shown in a two haplotype host-graft disparity between C57BL/6 recipients treated with either RmT1 or 30-H12 before challenging them with (BALB/c X AKR/J)F1 grafts, where the Thy-1.2 antigen concentration is approximately 50% compared to the density on BALB/c lymphocytes. Here, mAb 30-H12 loses its remarkable in vivo immunosuppressive quality, whereas RmT1 treatment protects mice against lethal GVHD. Binding sites were quantitated using a computerized approach for the analysis of data from ligand binding experiments of the respective mAb, RmT1 and 30-H12, coated to LN cells of BALB/c and F1 hybrid origin. Furthermore, the in vivo immunosuppressive activity of rat IgG2b antibodies directed against Thy-1 was found to correlate with their ability to generate stable antibody-C1q complexes on the cell surface of immunocompetent T cells.     

Leukaemia x fibroblast hybrid cells augment the antibody response to sheep red blood cells in inbred mice.

Spleen cells from mice undergoing a parasite-induced eosinophilia were fused with an azaguanine-resistant subline of the thymoma BW5147. A stable T hybrid (NIMP-TH1) was isolated and selected by recloning repeatedly by limiting dilution. The hybrid nature of NIMP-TH1 was confirmed by its expression of both parental alleles of Thy-1 and by chromosome analysis (modal chromosome number 102). On stimulation with phorbol myristate acetate, this hybrid releases a soluble activity which acts as a stimulator of eosinophil differentiation in vitro. Addition of hybrid conditioned medium to bone marrow cultures results in a selective stimulation of eosinophil production with no detectable increase in neutrophil or macrophage differentiation. The lymphokines interleukin-2 (IL-2) and interferon (IFN) are undetectable in NIMP-TH1 conditioned media. Although at high concentrations NIMP-TH1 supernatants are able to support very low levels of DNA synthesis in an IL-3-dependent cell line, and IL-3 appears to support low levels of eosinophil differentiation, dose-response curves show that the factor produced by NIMP-TH1 can be clearly segregated from IL-3 by its marked specificity for cells belonging to the eosinophil lineage. The factor present in these supernatants has been provisionally termed eosinophil differentiation factor (EDF).     

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.     

Detection of Thy-1 on cell surface of human T lymphoid cell lines by a monoclonal antibody

A mouse IgG2b(kappa) monoclonal antibody (MAb) HB-2S-1 against human brain Thy-1 was secreted by a hybridoma clone after fusion of mouse myeloma cells with spleen cells from a mouse that went through a prolonged immunization procedure before fusion. When tested against isolated human Thy-1 by the enzyme-linked immunosorbent assay (ELISA), MAb HB-2S-1 in culture supernatant showed a titer of over 100,000, and a titer of over 10 million in ascites of a mouse injected with the hybrid clone. By immunoblotting, this antibody was found to bind a doublet of protein bands of approximately 25,000 daltons among all proteins solubilized by deoxycholate (DOC) from membrane of human brain cells. When tested on human lymphoid cell lines by immunofluorescence, MAb HB-2S-1 strongly stained four T lymphoma cell lines, C91-Pl, HUT-102, HUT-78, and C5-MJ; and weakly two leukemia cell lines, MOLT-3 and Jurkat(clone E6-1). It did not stain a third T leukemia cell line, CCRF-CEM; a human B cell line, Raji; a plasmacytoma cell line, HMy2; or a myelomonocytic cell line, HL-60. Peripheral blood lymphocytes from ten normal human adults and the viable T cells isolated from another normal individual were also negative.     

Mouse/Human T-Cell Hybrids Rosetting with Sheep Erythrocytes

Hybrid cells have been recovered from selective culture medium after fusion of concanavalin-A-activated human lymphocytes with an AKR mouse thymoma (BW 5147). After 6 months of culture twenty-seven out of forty-nine clones still contained human chromosomes. Human chromosome 6 was present in 89% of these clones, and human X in 70%. Clones from one hybrid line contained several human chromosomes. In twelve of the clones carrying human chromosomes, the rosetting with sheep Erythrocytes (SRBC) was 3 times as high as In the BW 5147 cell line. All these clones Carried the human chromosome 6, and eight clones contained the human X chromosome as well. In some of these clones (25%) chromosome 6 was the only human one present. In the two clones In which human chromosome 6 was completely missing, the resetting with SRBC was at the level of the BW line. We therefore suggest that genes on human chromosome 6 are responsible for rosetting with SRBC.     

Enhanced expression of alkaline phosphatase in hybrids between neuroblastoma and embryonal carcinoma

Three independent hybrid cell lines were isolated from the fusion of clonal lines of embryonal carcinoma and neuroblastoma. A series of subclones was subsequently derived from the original hybrid clones. In early hybrid generations all hybrid lines showed enhancement of alkaline phosphatase activity, expressing 2–8 times the activity of the teratoma parental line. The overexpression of APase appears to take place in the stationary phase of the growth cycle. Segregation for very high levels of APase activity was observed among subclones of one hybrid line. Specific activities of the segregants ranged from 0.1 to 133. Results of heat denaturation studies are consistent with the hypothesis that it is the embryonal carcinoma APase that is being expressed in the hybrids.A preliminary account of this material has appeared (Reference 5). We thank Academic Press for permission to reprint material from that article.     

Monoclonal antibodies to xenotropic and MCF murine leukemia viruses derived during the graft-versus-host reaction

The humoral immune response during adult graft-versus-host reaction (GVHR) was assessed by the recovery of antibody-forming host spleen cells using cell fusion techniques. Two parent → F₁ strain combinations were studied, (B6 × D2)F₁ and (NFS × AKR)F₁ injected with the respective parental spleen cells. Hybridomas derived from recipient spleens were found to produce antibody with predominant specificity for murine leukemia virus (MuLV) envelope (env) polypeptides. The recovery of anti-MuLV monoclonal antibodies was dependent on the donor strain. Thus, D2 → (B6 × D2)F₁ and AKR → (NFS × AKR) resulted in a high incidence of anti-MuLV antibody production among the primary fusion products whereas no anti-MuLV hybridomas were recovered when B6 or NFS, respectively, were used as donors. Hybridomas derived from D2 → (B6 × D2)F₁ produced anti-MuLV antibodies of two general specificities: (1) broadly reactive, detecting determinants expressed by ecotropic and xenotropic MuLV, and (2) xenotropic MuLV specific. The latter group included antibodies reacting with all xenotropic MuLV and antibodies with xenotropic MuLV strain specificity. Xenotropic MuLV-specific determinants tere expressed by gp70, p15(E), and the gp70-p15(E) complex (gp90). This is to our knowledge, the first report of monoclonal antibodies specific for xenotropic MuLV. In contrast, hybridomas derived from AKR → (NFS × AKR) produced antibodies which reacted predominantly with unique determinants of a subgroup of MCF viruses. These results suggested that the anti-MuLV antibody repertoire expressed during GVHR is influenced by the endogenous MuLV of the respective mouse strain combination.     

Establishment and characterization of permanent murine hybridomas secreting monoclonal anti-thy-1 antibodies

hybridomas secreting anti-Thy-1 antibodies were produced by fusing cells of the mouse myeloma line P3-NSI/1-Ag4-1 (NS-1) with spleen cells from AKR/J mice immunized with C3H/Di thymus cells and by subsequent growth in tissue culture and selection of the hybrid cells. Two permanent hybridomas, 1B5 and 1aG4/C5, secreting antibodies of IgG3 subclass were isolated by repeated cloning of cells by dilution and in soft agar. Growth of the hybrid cell colonies depended on the presence of feeder cells; spleen cells at 1-2 x 10(6)/ml were most effective, then thymus cells at 1-4 x 10(6)/ml and peritoneal cells at a concentration 1-2 orders of magnitude lower. The two hybridomas were grown in vitro or vivo and their products were further analysed. In tissue culture in serum-free medium under the optimum conditions the supernatant from hybridoma 1B5 contained 0.07 mg/ml of antibodies and that from hybridoma IaG4/C5 had 0.26 mg/ml of antibodies, whereas ascites 1B5 contained 3.6 mg/ml and ascites 1aG4/C5 4.4 mg/ml of antibodies. A very low electrophoretic mobility of both antibodies facilitated their isolation. The specificity of the antibodies was tested in the cytotoxicity assay in the presence of complement and by the binding of isotopically labelled antibodies to thymus cells from A/Ph mice and other Thy-1.2+ strains and A.Thy-1.1 and AKR/J mie. Antibodies of clone 1aG4/C5 were specific for Thy-1.2+ cells, whereas antibodies of clone 1B5 at higher concentrations also reacted with Thy-1.1+ cells from the thymus and lymph nodes. Both antibodies killed more than 95% thymus cells and 60-70% lymph node cells in the cytotoxicity assay. The specificity of antibodies for T lymphocytes was confirmed in the functional test in which the antibodies eliminated the response of spleen cells to Concanavalin A but did not affect the response to lipopolysaccharide in the presence of complement.     

Supermelanotic hybrids derived from mouse melanomas and normal mouse cells

Hybrids formed between HPRT^– Cloudman mouse melanoma and normal cells were isolated. The parental origin of the hybrids was verified by isozyme and karyotype analyses. These hybrid cells differed in two major characteristics from hybrids of melanoma and established fibroblastic cells. (1) They grew as tumors when injected into mice, and (2) they expressed differentiated melanocytic functions. At least one of the differentiated functions was overexpressed. The specific activity of tyrosinase was 3–20 times higher in the hybrid cells than in the parental mouse melanoma. The overexpression of tyrosinase in these hybrid cells has been stable for more than a year, has been transmitted to subclones of the original hybrid cell lines, and has been expressed in tumors that grew after injections of hybrid cells into animals.     

T cell regulation of collagen-induced arthritis in mice. II. Immunomodulation of arthritis by cytotoxic T cell hybridomas specific for type II collagen

Injection of native type II collagen (CII) to susceptible strains of mice (H-2^q) induces a rheumatoid arthritis-like disease. To study the role of CD8⁺ T cells in the collagen-induced arthritis (CIA),we generated CII-specific T cell hybridomas by fusion of cells from arthritic C3H.Q mice and an AKR thymoma. Two hybrid clones (P3G8 and P2D9) were selected for their ability to lyse syngeneic CH-pulsed macrophages and recognize different antigenic epitopes in association with K^q molecules. When these T cell clones were irradiated and inoculated into (C3H.Q × AKR)F₁ mice 21 days prior to priming with native CII/ complete Freund's adjuvant, the incidence and the duration of CIA were significantly reduced in comparison to groups receiving saline or control T cell hybridoma. Furthermore, both anti-CII T cell hybridomas were able to attenuate CIA in highly susceptible inbred strains of mice and this suppression was antigen and disease specific. The protective activity seems to require intact cells as neither membrane fractions nor cytosolic preparations of the hybridoma T cells retained the vaccinating activity. Most importantly, one of the hybrid clones (P3G8) had a therapeutic effect on CIA since its administration to arthritic DBA/1 mice on day 30 after priming down-regulated the ongoing disease. Taken together, these findings suggest that anti-CII cytotoxic T cell clones can vaccinate against CIA and even reverse the disease.