Phenotypic characterization of mice of thymus target cells susceptible to productive infection by the radiation leukemia virus.

The spread of virus replication was studied by electron microscopy in the thymuses of inbred C57BL/Ka mice after intrathymic inoculation of the radiation leukemia virus (RadLV). The first type C-budding virus particles appeared in scarce blast cells of the subcapsular zone. Most of these blast cells were "X-cells," i.e., the thymus lymphoid cells most actively engaged in DNA synthesis. Virus replication spread to the entire cortical blast cell population and, from day 7 on, to the small cortical lymphocytes. The first virus-producing cells were derived from a very few target cells (approximately 0.001-0.003% of thymocytes) susceptible to RadLV infection. For determination of the phenotypes of these target cells, various thymocyte subpopulations obtained through a battery of cell separation methods were tested for their ability to support the replication of RadLV/VL3 virus in short-term culture. Most of these target cells were sensitive to the lytic effect of hydrocortisone and migrated in the fastest fraction of a 1Xg sedimentation gradient, together with the majority of [3H] thymidine-incorporating blast cells. They exhibited an intermediate density and expressed H-2 and Thy 1,2 cell surface antigens, although they were not found preferentially among the high Thy 1,2 population to which most of the cortical blast cells belonged. The spread of RadLV within the thymus and the surface phenotype characteristics of target cells indicate that these cells correspond to the thymocyte subset at the earliest stage of thymic lymphopoiesis and may be transitional between the prothymocytes and the subcapsular blast cell population.

Marrow-thymus interactions during radiation leukemogenesis in C57BL/Ka mice.

Transplantation of thymus and bone marrow cells from irradiated C57BL/Ka mice demonstrated the presence of potentially neoplastic cells in the thymus at 30 to 60 days postirradiation. During the same interval, no such cells could be detected in the bone marrow; moreover, the capacity of bone marrow cells to repopulate the thymus was impaired severely. These observations suggest that the primary site of neoplastic transformation in irradiated C57BL/Ka mice is the thymus rather than the bone marrow and that impaired thymic regeneration is a critical step in radiation leukemogenesis in mice.

Macrophage and lymphocyte-depleted thymus reticuloepithelial cell cultures: establishment and functional influence on T-lymphocyte maturation, C-type virus expression and lymphomatous transformation in vitro.

A method of cultivation has been developed that yields thymus reticuloepithelial (TRE) cell monolayers composed almost entirely of epithelial-like cells, with no detectable macrophages or lymphocytes. These cultures show the capacity to induce responsiveness to lectin mitogens in immature thymocytes. Such monolayers induce Thy-1 expression in nude mouse spleen and bone marrow cells. The functional macrophage-lymphocyte depleted TRE monolayers can be cryptically infected by radiation leukemia virus (RadLV). However, RadLV-infected TRE cells lack the capacity to induce neoplastic transformation in normal weanling and newborn thymocytes. Cultures prepared from C57BL/Ka mice treated with a leukemogenic course of irradiation produce a B-fibrotropic nonleukemogenic virus, and normal thymocytes cocultivated with these monolayers do not undergo in vitro transformation. It is concluded that TRE monolayers depleted of macrophages and lymphocytes: (1) exhibit the functional capacity to induce some of the properties of mature T cells; (2) are infectable by RadLV; (3) are one of the sites for expression of ecotropic virus after leukemogenic fractionated irradiation; and (4) are not capable of transforming normal thymocytes in vitro.

Cytotypically specific transfecting activity of DNA from C57BL/Ka mouse cells producing thymotropic in contrast to nonthymotropic retroviruses.

A comparative study has been made of the susceptibility of fibroblastic cells to transfection by DNA from C57BL/Ka mouse lines producing either fibrotropic or thymotropic retroviruses. DNA isolated from fibroblasts that release a B-ecotropic, fibrotropic virus, BL/Ka(B), was found to transfect fibroblasts of Fv-1bb genotype with release of virus similar to BL/Ka(B). Fv-1nn fibroblasts were also susceptible but expression was delayed, and xenotypic mink lung cells were refractory. In contrast, DNA prepared from a murine T-cell lymphoma line producing a B-ecotropic, thymotropic virus failed to transfect mouse fibroblasts though it transfected a nonproducer T-cell lymphoma line. The data suggest that the Fv-1 and differentiation-specific restriction mechanisms operate at different molecular levels.

Detection of infectious centers in C57BL/Ka lymphoid cell populations infected in vitro by the radiation leukemia virus.

The cocultivation of nonproducer lymphoma cells derived from a radiation-induced lymphoma of the C57BL/Ka mouse with cultures of lymphoid cell populations from the thymus, spleen, and marrow of the same strain 48 hr after their infection by the C57BL/Ka leukemia viruses permits the detection of infectious centers in these cultures. A quantitative assay is described which allows the estimation in lymphoid cell subpopulations of the numbers of target cells susceptible to productive infection by the thymotropic and leukemogenic viruses of C57BL/Ka mice in vitro. This assay should greatly facilitate the identification and characterization of such target cells.

In vivo infectivity of the fibrotropic C-type viral isolates from C57BL/Ka mice.

Of the three fibrotropic C-type viral isolates from C57BL/Ka mice, only the BL/Ka(B) virus is capable of infecting normal hematopoietic and lymphoid cell populations of C57BL/Ka mice in vivo, and none are tumorigenic. Inoculation of this virus alone into neonates resulted in transient replication in the bone marrow, spleen, and occasionally the thymus. Thymocytes could, however, be permanently infected in such animals if BL/Ka(B) were coinoculated with the xenotropic BL/Ka(X) virus. Neonatal injection of BL/Ka(B) prior to fractionated wholebody irradiation yielded an increase in the percentage of virus-productive radiogenic lymphomas and a decrease in incidence of such tumors. Injection of BL/Ka(B) into normal adult C57BL/Ka mice did not yield overt expression of virus replication in any of the tissues tested; latent infection could, however, be detected in the marrow and in the reticuloepithelium of the thymus. Whole-body X-irradiation of adults with 400 rads partially restored the neonatal susceptibility of bone marrow cells to infection by the isolate. BL/Ka(B) injection after fractionated whole-body irradiation of weanling C57BL/Ka mice increased the percentage of virus-positive lymphomas and revealed that a bone marrow cell subpopulation permissive for infection by the virus increases greatly in abundance soon after irradiation.

Establishment, characterization and virus expression of cell lines derived from radiation- and virus-induced lymphomas of C57BL/Ka mice.

Permanent cell lines have been established in vitro from lymphoid tumors induced in C57BH/Ka mice by fractionated X-irradiation or by inoculation of the radiation leukemia virus (RadOV). The cultured cells are lymphoblastic, replicate rapidly in vitro, and are tumorigenic in vivo. The cell surface markers Thy 1, Ly 1, Ly 2,3 and GIX are expressed by the lymphoid tumor cells in the mouse and persist in the corresponding cell lines; expression of the H-2 and TL antigens is greatly reduced during in vitro passage, but is restored on in vivo transplantation. The cell lines derived from RadLV-induced tumors (BL/VL lines) produce a virus population (RadLV/LTC) with the thymotropic and leukemogenic attributes of RadLV. Those derived from radiation-induced, virus-negative lymphomas (BL/RL lines) are initially devoid of MuLV expression, but frequently become spontaneous virus producers during in vitro cultivation.

Specificity of cell surface virus receptors on radiation leukemia virus and radiation-induced thymic lymphomas.

We have developed a system for analysis of murine leukemic virus (MuLV) receptors on the surface of thymic lymphoma cells utilizing the fluorescence-activated cell sorter. The binding of fluoresceinated or rhodaminated MuLV to target cells showed saturation kinetics and was blocked by homologous MuLV, and bound MuLV had a polypeptide profile identical to that of input MuLV. Thymic lymphomas bound specifically the MuLV which induced them, whereas only 0.5 to 2% of normal thymocytes showed equivalent MuLV binding. Simultaneous binding of excess fluoresceinated RadLV and rhodaminated MCF-247 AKR virus to radiation leukemia virus-induced or spontaneous AKR thymic lymphomas demonstrated that even in the presence of both viruses the cells bound preferentially the inducing MuLV. Examination of the C57BL/Ka endogenous viruses showed that radiation leukemia virus-induced thymic lymphomas bind only thymotropic-leukemogenic radiation leukemia virus and not eco- or xenofibrotropic MuLV's. Thus, virus binding in this system involves only leukemogenic isolates of these retroviruses and implies a central role of this receptor-ligand interaction in the processes of leukemic transformation.

Serological characterization of B-tropic viruses of C57BL mice: possible origin by recombination of endogenous N-tropic and xenotropic viruses.

The serological properties of the gag gene products p15 and p12 of N- and B-tropic viruses of C57BL mice have been examined. Although these viruses were serologically identical by competition assays for proteins gp71 and p30, they were readily distinguishable in competition assays for proteins p15 and p12. Two isolates of N-tropic viruses had p12s serologically indistinguishable from AKR murine leukemia virus p12, while two B-tropic isolates had distinctly different p12s. The latter p12s were serologically indistinguishable from the p12 purified from the B-tropic radiation leukemia virus (RadLV)/VL-3. Moreover, this p12 was indistinguishable from the p12 of the endogenous C57BL/Ka xenotropic virus. Similarly, the p15s of the B-tropic viruses were serologically distinct from the AKR murine leukemia virus type of p15, as was the p15 of one C57BL N-tropic virus, whilc another N-tropic isolate had a p15 identical to the AKR murine leukemia virus p15. These results are interprered to suggest that the endogenous N-tropic virus of C57BL mice undergoes recombination with the endogenous, xenotropic virus and that this mechanism is involved in the generation of B-tropic viruses in C57BL mice.

Macrophage-mediated in vitro sensitization of lymphocytes. II. The detection of neo-antigens on transformed lymphocytes and passages of normal fibroblasts.

Unprimed lymphocytes were sensitized in vitro by incubating them with syngeneic macrophages that had been fed with viral or cellular antigens. The sensitized lymphocytes were tested for their cytotoxic activity against virus-infected and noninfected fibroblasts. The antigenic preparations used for priming the macrophages were either tumor cell-free extracts or supernatants from virus productive cells. Cell-free extracts from the productive RadLV-induced lymphoma cells or the nonproductive radiation-induced lymphoma cells were immunogenic when presented to lymphocytes by macrophages. In contrast, cell-free extracts from normal thymocytes were much less immunogenic, suggesting that the presence of viral associated antigens (VAA) can selectively be detected on lymphoma cells by this assay. Fibroblastic cell lines but not primary fibroblasts were also susceptible to the cytotoxic lymphocytes induced by RadLV-fed macrophages. Primary fibroblasts became susceptible to the sensitized lymphocytes either after infection with the corresponding virus, or if not infected, after several passages in vitro, suggesting that neo-antigens cross-reacting with viral antigens appear during sub-culturing of fibroblasts in vitro. This system makes it possible to detect VAA either as immunogens when presented to lymphocytes by macrophages, or as targets for cytotoxic lymphocytes.

Isolation of a type C RNA virus from an established human histiocytic lymphoma cell line.

A type C RNA virus has been detected in the culture fluids of the SU-DHL-1 human histiocytic lymphoma cell line previously established in this laboratory. In electron micrographs, the virus closely resembled other typical mammalian type C RNA tumor viruses in size and morphology. Viral RNA-dependent DNA polymerase activity has been demonstrated in particles (densities of 1.15 and 1.22 g/ml) in the microsomal cytoplasmic fraction and in pellets of culture fluids. The enzyme is partially inhibited by antibodies to the RNA-dependent DNA polymerases of simian sarcoma virus and RD-114 virus but not by antibody to the polymerase of murine leukemia virus, suggesting some degree of relatedness to type C viruses of subhuman primate origin. Typical syncytial microplaques were induced when SU-DHL-1 cells were cocultivated with rat XC cells. Although no focus formation was noted in similarly cocultivated mouse UC1-B cell cultures, the numbers of foci induced in rat embryo fibroblasts by murine sarcoma virus were significantly increased by coinfection with the virus from SU-DHL-1 cell culture fluids. No other evidence of infectivity, inducibility, or capacity for helper rescue of defective murine sarcoma virus genomes has been detected to date in cocultivation studies with a spectrum of fibroblastic and other nonlymphoid indicator cell lines of human and other species of origin.