Thymic Lymphoma Cell Line Research Articles

Characterization of rat CD80 and CD86 by molecular cloning and mAb.

The CD28/B7 pathway provides a critical co-stimulatory signal for T cell activation. In the present study, we cloned rat CD80 and CD86 cDNA from a HTLV-1-transformed rat T cell line, Lewis-S1, expressing a high level of CTLA-4-Ig binding proteins. The predicted CD80 and CD86 polypeptides were composed of 321 and 313 amino acids respectively, and exhibited features common to human and mouse B7 family proteins. Both CD80 and CD86 mRNAs were abundantly detected in HTLV-1-transformed rat T cell lines but not in a thymic lymphoma cell line. To further explore the function of rat CD80 and CD86, we generated cDNA transfectants and anti-rat CD80 (3H5) and anti-rat CD86 (24F) mAb. Rat CD80 or CD86 transfectants exhibited a potent co-stimulatory activity for rat T cell proliferation, which was blocked by 3H5 and 24F mAb respectively. 3H5 or 24F immunoprecipitated a 80-90 or 90-100 kDa surface protein from Lewis-S1 cells. HTLV-1-transformed rat T cell lines expressed high levels of both CD80 and CD86 as estimated by staining with 3H5 and 24F, which acted co-stimulatory for allogeneic T cell activation as estimated by blocking with 3H5 and 24F. These mAb will be useful for investigating the pathophysiological functions of CD80 and CD86 in transplantation, autoimmune diseases and HTLV-1-associated pathologies in the rat system.

I-309/T cell activation gene-3 chemokine protects murine T cell lymphomas against dexamethasone-induced apoptosis.

We have previously reported that cytokines such as IL-9, IL-4, and IL-6 protect murine thymic lymphoma cell lines against dexamethasone-induced apoptosis. A similar activity, which could not be ascribed to any of these factors, was found in a number of human T cell supernatants that enabled mouse BW5147 thymic lymphoma not only to escape apoptosis but also to maintain proliferation. The protein responsible for this activity was purified to homogeneity from the culture medium of activated leukemic T cells and was found to be identical with the I-309 chemokine. Half-maximal anti-apoptotic activity was obtained with approximately 1 ng/ml, a concentration considerably lower than that required for the monocyte chemotactic activity of this molecule, as measured on THP-1 cells. The purified I-309 also improved the survival of two other mouse thymic lymphoma cell lines. This activity was as potent as that of IL-9, which was the strongest anti-apoptotic factor found to date for these cells. Similar results were obtained for BW5147 cells with recombinant I-309 and with T cell activation gene-3, the murine homologue of I-309, but not with other members of the chemokine family, including IL-8, neutrophil-activating peptide-2, granulocyte chemotactic protein-2, macrophage inflammatory protein-1a, RANTES (regulated upon activation, normal T cell expressed and secreted), monocyte chemotactic protein-1 (MCP-1), and MCP-2. MCP-3, however, showed a minor, but significant effect in this model. Unlike that of IL-9, the activity of I-309 was completely inhibited in the presence of pertussis toxin, indicating the involvement of a G protein in this process.

In vitro maturation of clonal CD4+CD8+ cell lines in response to TCR engagement.

Engagement of the TCR on immature CD4+CD8+ (DP) thymocytes by an appropriate peptide/MHC ligand evokes a complex program of maturation known as positive selection. As a result, DP thymocytes are rescued from programmed cell death, become committed to the CD4 or CD8 lineage, extinguish expression of V(D)J recombinase activity, and undergo further maturation. We describe here a panel of DP thymic lymphoma cell lines that, in response to in vitro TCR engagement, undergo many of the TCR-beta-induced maturation events that have been reported to accompany positive selection of DP thymocytes in vivo. These events include increased expression of CD5, CD69, CD45, TCR-alpha, and MHC class I, and decreased expression of Thy-1 and heat-stable Ag. In addition, we observed TCR-induced expression of the bcl-2 gene, a well described inhibitor of programmed cell death. Finally, TCR engagement decreased expression of recombinase-activating genes and terminal deoxynucleotidal transferase genes, as well as V(D)J recombinase activity. However, TCR engagement did not elicit demonstrable CD4/CD8 lineage commitment. These observations suggest that engagement of the TCR on these DP cell lines elicits multiple maturation events that are part of the positive selection developmental program, but not CD4/CD8 lineage commitment. Thus, these DP cell lines provide the opportunity to elucidate molecular mechanisms of maturation and CD4/CD8 lineage commitment in vitro.

Interspecies somatic T cell hybrids as biological tools for studying gene expression during T cell development.

Interspecies somatic cell hybrids were generated by fusing the mouse thymic lymphoma cell line, BW5147, with normal human T lymphocytes at different stages of differentiation. Thymocytes, activated peripheral T lymphocytes, or an activated T cell clone were used as human partners, respectively, in three independent fusions. Phenotype and genetic analysis demonstrated that these hybrids preferentially segregated human chromosomes while retaining a complete mouse genetic complement, irrespective of the human partner used for fusion. A large number of T cell differentiation antigens constitutively expressed throughout the T lymphocyte development remained constitutively expressed in the hybrids, irrespective of the maturation stage of human partner used for fusion. In contrast, the expression of other antigens related to a specific stage of T cell development (CD2, CD8), or to an activated state of T lymphocytes (HLA-DR, CD25), was to observed in the hybrids, with no apparent correlation with the segregation of human chromosomes other than, of course, the encoding chromosome. From these results we suggest that the developmental stage of the fusion partners strongly influences the pattern of expression by activating or silencing genes programmed to be expressed in distinct phases of T cell ontogeny.

Cellular depletion of p56lck during thymocyte apoptosis

The src-related protein tyrosine kinase p56lck is thought to be important in regulating maturation and functional responsiveness of T cells and thymocytes. In the present studies we report that expression of p56lck is suppressed during apoptosis. Using primary cultures of rat thymocytes, we found that agents that are effective in inducing apoptosis, including okadaic acid, dexamethasone, and antibodies to the CD3 receptor, also deplete cells of p56lck. This process is rapid, occurring within 24 h, and is not due to cytotoxicity. Inhibition of DNA fragmentation in apoptotic cells with the endonuclease inhibitor ZnCl2 failed to prevent depletion of p56lck, suggesting that it was not a consequence of the DNA degradation process. Using the thymic lymphoma cell line LSTRA, apoptosis was also associated with cellular depletion of p56lck. In contrast to thymocytes, this process required 48-72 h possibly because these cells overexpress p56lck. Although at this time we are uncertain as to the precise role of p56lck in the process of apoptosis, our results indicate that changes in the expression of this protein in thymocytes is an important marker of programmed cell death.

Identification of a 16-kDa thymocyte membrane glycoprotein involved in the thymocyte/thymic medullary epithelial cell interaction.

We have previously described a type of lymphoepithelial interaction involving CD4+ CD8+ thymocytes and a medullary epithelial cell line (E-5). This interaction is mediated by the recently described gp23/45 epithelial adhesion molecule and an as yet unknown thymocyte receptor. The present work describes a thymocyte surface glycoprotein of 16 kDa which binds both to E-5 cells and to the purified gp23/45 adhesion molecule. In addition, a thymic lymphoma cell line (Ti-6), which interacts with the E-5 cells via the gp23/45 receptor, also present a 16-kDa glycoprotein on its surface. Taken together, the data suggest that the 16-kDa thymocyte surface glycoprotein participates in the binding between these cells and the thymic epithelium.

Thymic lymphomas mediate non-MHC-restricted, TNF-dependent lysis of the murine sarcoma WEHI-164

Tumor necrosis factor (TNF), lyses a range of sensitive tumor targets and has been shown to be the mediator of natural cytotoxic (NC) activity first described in our laboratory. In this report, we identify two thymic lymphoma cell lines which lyse the prototype NC target WEHI-164 and share characteristics of NC. R1.1E and L5178-27av lyse the WEHI-164 sarcoma in 18-hr 51Cr release assays via a TNF-dependent, non-MHC-restricted (R1.1E) mechanism although they do not constitutively produce TNF. NC- and TNF-resistant variants of WEHI-164 are resistant to lymphoma-mediated lysis. Expression of the ganglioside GD3 by the lymphomas correlates with their relative levels of lysis. Thus, GD3, which is known to have a role in T cell activation may be involved in recognition or triggering for TNF-dependent cell-mediated lysis.

Thymic lymphoma cells as a model for complex-formation between thymocytes and thymic medullary epithelial cells

The formation of complexes between thymocytes and thymic stromal elements is known to be involved in T cell differentiation. We have previously described one type of lympho-stromal interaction involving CD4 +CD8 + thymocytes and a medullary epithelial cell line (E-5). In this study we report the potential for complex formation of two different thymic lymphoma cell lines (Ti-6 and RDM-4), Ti-6 cells were shown to adhere to the E-5 cells, while RDM-4 cells were totally incompetent. Phenotypic characterization of these cell lines suggests that immature thymocytes are not capable of forming complexes with medullary epithelium and that a certain level of differentiation is required to do so. Comparison of their phenotypes showed that the possibility of some classical T cell surface markers being the receptor for the E-5 ligand can be dismissed.

Mouse thymoma cell line expresses a gluconeogenic enzyme, fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase was observed in a thymic lymphoma cell line, WEHI7.1 (11.5 ± 0.8 munits/mg cytosol protein). Only a trace amount of the enzyme activity was observed in normal thymus tissue. The WEHI7.1 enzyme had a pH optimum at around 7.5. The AMP-concentration giving 50% inhibition of the activity was about 73 μ M. That of the crude mouse liver enzyme was 35 μ M. The antibodies against the liver and intestinal enzymes cross-reacted with the WEHI7.1 enzyme with a lower affinity than the liver enzyme. Immunoblot showed that the subunit molecular weight of the WEHI7.1 enzyme was the same as that of the liver enzyme.

Molecular biological characterization of a highly leukaemogenic virus isolated from the mouse. IV. Viral proteins.

The proteins of a highly leukaemogenic murine virus (DMBA-LV) endogenous to the CFW/D mouse have been characterized. This virus does not contain ecotropic, xenotropic or polytropic type C retroviruses capable of replicating in established tissue culture cell lines. Analysis of the viral proteins indicated that proteins characteristic of both type C and type B retroviruses were present. All the proteins characteristic of a competent, mature type B retrovirus, such as the murine mammary tumour virus, were present in the virions and were expressed at high levels in a virus-producing thymic lymphoma cell line. Type C internal structural proteins were detected in the virus and cells producing virus. Type C envelope protein was detected at very low levels in both the virions and the virus-producing tumour cell lines. Chymotryptic polypeptide profiles of this gp70 indicated similarity with the envelope glycoproteins characteristic of xenotropic and recombinant type C retroviruses.

Methylation of deoxycytidine in replicating cells treated with ultraviolet radiation and chemical carcinogens.

5-Methyl-2'-deoxycytidine (m5dC) levels were measured in DNA from three types of cultured cells following treatment with u.v. radiation and two chemical carcinogens, N-methyl-N-nitrosourea (MNU) and N-acetoxy-2-acetylaminofluorene (NA-AAF). Control values for m5dC in Raji cells (a human lymphoblastoid cell line), S49 cells (a mouse thymic lymphoma cell line) and human diploid fibroblasts are 3.6%, 3.6% and 3.2%, respectively. None of the damaging agents produced a detectable change in methylation levels of newly replicated DNA, even at levels of damage that inhibited replication by 95%. In contrast, treatment with 5-aza-2'-deoxycytidine, a known methyltransferase inhibitor, transiently reduced genomic methylation by 89% and 74% in Raji and S49 cells, respectively. Although other investigators have found a marked reduction in m5dC in DNA replicated after carcinogen treatment, our experiments indicate that extensive demethylation is not a necessary consequence of DNA damage.

Molecular biological characterization of a highly leukaemogenic virus isolated from the mouse. III. Identity with mouse mammary tumour virus.

A highly leukaemogenic virus isolate (DMBA-LV) endogenous to the CFW/D mouse has been found to contain two viral genomes. One was closely related to the type B milk-borne mouse mammary tumour virus (MMTV) and present in tenfold excess over a type C viral genome which was only partially related to xenotropic and polytropic isolates from the CFW/D mouse as well as to the ecotropic Moloney murine leukaemia virus isolate. The thymic lymphoma cell line that produced DMBA-LV expressed high levels of MMTV viral RNA (35S and the 24S envelope mRNA). Both the virus and the virus-producing cell line expressed multiple species of type C viral RNA. Similar species of type C viral RNA were also associated with non-infectious, non-leukaemogenic viral particles present in both normal lymphoid cells and in a MMTV-free thymic lymphoma cell line established from a second chemical carcinogen-induced tumour.

Provisional chromosome assignment of the rat major histocompatibility complex.

Somatic cell hybrids between an AKR mouse thymic lymphoma cell line and normal F344 rat spleen cells were isolated in order to assign the rat major histocompatibility complex (MHC) (RT1) to a specific chromosome. Six primary hybrid clones and 3 of their derivatives were examined for expression of the RT1 antigens on the cell surface by the complement-dependent cytotoxicity test and simultaneously for their karyotypes by the Hoechst 33258/quinacrine mustard double staining method. All of these hybrid clones consisted of 2 sets of mouse and 1 set of rat parental cells, with a preferential loss of rat chromosomes. A high degree of concordance was shown between the expression of the RT1 antigen and the presence or absence of rat chromosome 14 in the original 6 hybrid clones. This was confirmed in 1 spontaneous and 2 immunoselected RT1-negative segregants from RT1-positive clones. These results led us to propose that the rat MHC is located on chromosome 14.

Impaired Maturation of Mouse Mammary Tumor Virus Precursor Polypeptides in Lymphoid Leukemia Cells, Producing Intracytoplasmic A Particles and No Extracellular B-Type Virions

Processing of polypeptides of the mouse mammary tumor virus, a type B retrovirus, was investigated in a transplanted thymic lymphoma cell line of the GR strain (GRSL). This cell line was maintained in vivo in ascites form and in vitro as a suspension culture. GRSL cells produce clusters of intracytoplasmic A particles and are virtually deficient in the production of mature extracellular B-type particles. As control, a mammary tumor cell line of the same mouse strain capable of complete virion synthesis was used. The kinetics of viral polypeptide synthesis were studied by pulse labeling with various isotopes (including (35)S and (32)P), followed by immunoprecipitation of cell lysates with monospecific antisera to the major mouse mammary tumor virus gag and env proteins, p27 and gp52, respectively. Both the primary gag and env precursor polypeptides were synthesized in the GRSL cells, but their conversion into viral proteins was impaired. The major gag precursor, Pr73(gag), was stable over a period of 8 h, and mature viral core polypeptides could not be detected. Also, the highly phosphorylated intermediates in the proteolytic processing of Pr73(gag) in virus-producing cells were absent in GRSL cells. By immunoprecipitation, Pr73(gag) was detected in a GRSL particle fraction with the density of intracytoplasmic A particles. The precursor for envelope proteins, Pr73(env), was turned over without the generation of mature viral envelope components gp52 and gp36. The in vivo-transplanted ascites GRSL cells, however, were shown to express gp52 on the cell surface together with a 73,000-dalton polypeptide, as indicated by cell surface iodination and immunoprecipitation.

Evidence that mulv‐induced thymic lymphoma cells possess specific cell membrane binding sites for mulv

Cell-surface binding sites specific for thymotropic murine leukemia viruses were found in high concentrations on thymic lymphoma cell lines induced by this class of virus, but were detectable in much lower concentrations (if at all) in several non-T leukemias, plasmacytomas, and normal thymocytes or spleen cells. By specific comparison, Moloney leukemia virus (M-MuLV) binds to a lymphoma induced by M-MuLV, but not to a thymic lymphoma induced by Gross leukemia virus (G-MuLV); and G-MuLV binds to an AKR lymphoma but not to the M-MuLV-induced lymphoma. The material which binds to these T-lymphoma membrane sites is input virus, rather than a contaminant which copurifies with virus. Autoradiographic analysis demonstrates that a high proportion of T-lymphoma cells possess binding sites, whereas only a rare cell in the thymus binds murine leukemia virus to the same degree. We raise and discuss the hypothesis that each T lymphoma induced by thymotropic leukemia viruses may represent the clonal descendants of the few rate cells in the normal thymocyte population which also bind these viruses.