ABSTRACT The murine retrovirus SL3-3 causes malignant transformation of thymocytes and thymic lymphoma in mice of the AKR and NFS strains when they are inoculated neonatally. The objective of the present study was to identify the primary target cells for the virus in the thymuses of these mice. Immunohistochemical studies of the thymus after neonatal inoculation of the SL3-3 virus showed that cells expressing the viral envelope glycoprotein (gp70 + cells) were first seen at 2 weeks of age. These virus-expressing cells were found in the cortex and at the corticomedullary junction in both mouse strains. The gp70 + cells had the morphology and immunophenotype of dendritic cells. They lacked macrophage-specific antigens. Cell separation studies showed that bright gp70 + cells were detected in a fraction enriched for dendritic cells. At 3 weeks of age, macrophages also expressed gp70. At that time, both gp70 + dendritic cells and macrophages were found at the corticomedullary junction and in foci in the thymic cortex. At no time during this 3-week period was the virus expressed in cortical and medullary epithelial cells or in thymic lymphoid cells. Infectious cell center assays indicated that cells expressing infectious virus were present in small numbers at 2 weeks after inoculation but increased at 5 weeks of age by several orders of magnitude, indicating virus spread to the thymic lymphoid cells. Thus, at 2 weeks after neonatal inoculation of SL3-3, thymic dendritic cells are the first cells to express the virus. At 3 weeks of age, macrophages also express the virus. In subsequent weeks, the virus spreads to the thymocytes. This pathway of virus expression in the thymus allows the inevitable provirus integration in a thymocyte that results in a clonal lymphoma.

Objective Understanding the interaction between HIV and developing thymocytes is crucial in determining how HIV infection perturbs the immune system. We determined which thymocyte subsets can harbor and express HIV. Design HIV expression in mature and immature thymocytes obtained from surgical specimens from non-infected children was determined after in vitro infection with the syncytium-inducing, cytopathic NL4–3 and the non-syncytium-inducing, relatively noncytopathic JR-CSF isolates. Methods Intracellular staining for the HIV p24gag antigen was combined with cell surface phenotyping to determine thymocyte subsets expressing HIV. Infection was quantitated by polymerase chain reaction on sorted subsets. Results NL4–3 replicated faster and to higher titers and caused a more severe decrease of all CD4-bearing thymocytes than did JR-CSF. In addition, both immature CD1+ and mature CD1− thymocytes expressed NL4–3, whereas only mature CD1 -cells expressed JR-CSF. The tropism of NL4–3 for these immature cells suggests a mechanism for a more profound impact on T-cell maturation than that seen with JR-CSF. We also found that thymocytes lacking cell surface CD4 (CD4-CD8− and CD4-CD8+ subsets) expressed virus with either isolate late in infection, when viral levels were high. The CD4-CD8− cells expressing HIV were mature CD3bright T-cell receptor (TCR)α/βbright cells. Conclusions These results show that NL4–3 can be expressed by thymocytes at immature and mature stages of differentiation and cause severe loss of CD4+ cells. Thus, tropism of a virus for immature cells can affect the capability of the thymus to produce new T lymphocytes leading to a greater impact on development and functions of the immune system. It is proposed that this in vitro model can be used to study pathogenic mechanisms in the thymus.

The thymus is essential for normal T cell development and is particularly active during fetal and postnatal life. Here we describe in vitro studies of HIV-infected thymocytes cultured with cytokines normally produced in the thymus. Virus expression was determined by measuring p24 antigen levels in the culture supernatants. Addition of IL-2+IL-4 and IL-4+IL-7 to the HIV-infected cultures of both fetal and postnatal thymocytes resulted in various levels of synergistic expression of p24 antigen. When differences in phenotype between HIV-infected and non-infected (sham-treated) cultures from the same specimen were evaluated, there was a decrease in the percentages and absolute numbers of CD4-bearing cells in HIV-infected thymocytes cultured with IL-2+IL-4. Studies were done to determine if synergy in HIV expression was mediated by activation, proliferation or induction or suppression of other cytokines. We found a higher percentage of activated CD4(+)CD8(+)/(high) cells in thymocytes cultured with IL-2+IL-4 and IL-4+IL-7 than in thymocytes cultured with IL-2+IL-7. Proliferation was higher in thymocytes cultured with cytokine combinations but did not correlate with those conditions showing synergy. IL-4 reduced IFN-gamma production by thymocytes cultured with IL-2 in both HIV-infected and non-infected thymocytes. In addition, exogenous IFN-gamma decreased p24 expression by HIV-infected thymocytes when cultured with IL-4 alone, with IL-2+IL-4 or IL-4+IL-7. These results suggest that suppression of IFN-gamma by IL-4 may combine with cell activation and proliferation to produce synergy of virus expression observed with IL-2+IL-4 and IL-4+IL-7.

We have identified nucleotide sequences that regulate transcription in both a cell-type-specific and general manner in the long terminal repeat of the MCF13 murine leukemia virus. Besides the enhancer element, we have observed that the region between the enhancer and promoter (DEN) has a profound effect on transcription in different cell types. This effect, however, was dependent on the copy number of enhancer repeats and was detectable in the presence of a single repeat. When two enhancer repeats were present, the effect of DEN on transcription was abrogated except in T cells. DEN also makes a significant contribution to the leukemogenic property of the MCF13 retrovirus. Its deletion from the MCF13 virus dramatically reduced the incidence of thymic lymphoma and increased the latency of disease in comparison with the wild-type virus. This effect was most marked when one rather than two enhancer repeats was present in the mutant viruses. We also observed that the removal of one repeat alone remarkably reduced leukemogenicity by the MCF13 virus. A newly identified protein-binding site (MLPal) located within DEN affects transcription only in T cells, and its deletion attenuates the ability of an MCF13 virus with a single enhancer repeat to induce thymic lymphoma. This observation suggests that the MLPal protein-binding site contributes to the effect of the DEN region on T-cell-specific transcription and viral leukemogenicity. This study identifies the importance of nonenhancer sequences in the long terminal repeat for the oncogenesis of the MCF13 retrovirus.

These studies were designed to look for a correlation of intrathymic survival of virus-infected thymocytes with lymphomagenesis. Cells from the normal-appearing prelymphoma thymus of SL3-3 virus-treated AKR mice were studied. Also, phenotypic properties of the malignant cells from the virus-induced lymphomas are described. In this model system, 100% of mice inoculated with virus at three days of age develop thymic lymphoma between 60 and 90 days of age. The experiments show that cells with malignant potential do not appear in the thymus until 36 days after virus inoculation. These cells are initially thymus-dependent (TD) in that they produce lymphoma of donor-type in recipients after intrathymic inoculation with long latency. They do not produce lymphoma after subcutaneous inoculation in syngeneic hosts. At 39 days after virus inoculation, the first thymus independent (TI) lymphoma cells appear. These cells, like the cells isolated from thymi with overt tumors, produce lymphoma of donor-type after a latency when inoculated by the intrathymic or subcutaneous route. Thymocytes from normal-appearing thymi of mice at 42 days after virus inoculation, which could be expected to include TD, TI or no lymphoma cells, were evaluated for their ability to survive in a recipient thymus for three weeks after intrathymic inoculation. They were compared to thymocytes from age-matched control mice. Thymi receiving the virus-infected thymocytes showed 15% to 80% donor cells at three weeks. The highest numbers of donor cells were from thymi which were shown to contain TI lymphoma cells. However, cells from thymi with TD and no lymphoma cells could also be detected in significant numbers at three weeks after intrathymic inoculation. Less than 2% of donor-type thymocytes could be found after inoculation of thymocytes from normal control AKR mice. These data provide evidence that virus infection of thymocytes, even before the appearance of cells with lymphomagenic potential, endows them with a capacity for prolonged intrathymic survival. This appears to be a necessary step for tumor progression in this model. A remarkable phenotypic diversity of the virus-induced lymphomas was shown. The effect of various growth environments, intrathymic, subcutaneous, and in vitro on lymphoma cell phenotypic expression revealed individual differences in each tumor and in each environment.

These studies report changes occurring in the thymus of AKR and NFS/N mice after infection with the lymphomagenic retrovirus SL3-3. In virus-infected AKR fetal thymus, the programmed cell death caused by treatment with antibody to CD3 was remarkably diminished. A method of establishing thymic stromal cultures from mice of 1 to 3 wk of age is described. Using this method, it was found that SL3-3 virus infection by neonatal inoculation allowed establishment of thymic stromal cultures from organs removed from AKR mice of 30 to 50 days of age and from lymphomas, whereas thymic stromal cultures could not be established from control mice after 30 days of age. Using NFS/N mice which have no endogenous virus, it was shown that infection of thymic stroma precedes infection of thymocytes and that thymocytes are permissive for infection with SL3-3 virus but not for the nononcogenic retrovirus, Akv, yet Akv virus replicates efficiently in thymic stroma. SL3-3 virus integrates randomly in each lymphoma induced by this virus. The lymphomas are clonal or oligoclonal. Pim-1 and c-myc genes commonly rearranged in other virus-induced thymic lymphoma showed rearrangement in only a few lymphomas. A theory is proposed, based on the work presented here and in recent studies, which states that SL3-3 virus infection of thymic stroma allows infection of thymocyte progenitors entering from the bone marrow. These cells are then altered so that their maturation is delayed and their intrathymic survival is prolonged. This permits virus integration and reintegration that results in the genetic changes which transform the cell.

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A chronological study of the individual thymic lobes of young AKR mice after neonatal inoculation of the oncogenic AKR retrovirus SL 3-3 was performed. 100% of mice treated in this manner develop lymphoma between 60 and 100 days of age. A search for early lymphoma cells in individual thymi was carried out by inoculating the thymocytes subcutaneously in syngeneic and intrathymically in syngeneic and semisyngeneic recipients. Tumor progression was observed in animals between 48 and 60 days of age. These animals have: (a) normal weight lobes, in which no lymphoma cells could be detected, (b) thymus-dependent lymphoma cells, in one or both normal weight lobes; (c) thymus-independent lymphoma cells, found in lobes of normal weight as well as in thymi enlarged by lymphoma cells. Thymocyte characteristics of virus-treated animals of 21 to 63 days of age were compared with those of age-matched controls. Beginning at 28 days a concordant, progressive with time, increase of thymocyte surface staining for the viral envelope glycoprotein gp70 was seen in all lobes from virus-treated animals. Evaluation of cell surface markers by two-color fluorescence with antibodies to CD4 and CD8 showed that after 50 days of age, thymic lobes with and without lymphomas had nonspecific, but marked, alterations of the typical thymocyte surface marker pattern. No characteristic CD4, CD8 surface phenotype was found in primary lymphomas. Using probes for the T-cell receptor J beta 2 gene segments and the Akv ecotropic virus gp70 envelope genes, oligoclonality in J beta 2 rearrangements and clonality using the Akv env genes was demonstrated in thymi with the thymus-dependent phenotype. In lymphomas T-cell receptor beta gene probes showed either oligoclonality or clonality. Clonal virus integrations were found in these lymphomas. These experiments suggest the following series of events in virus-accelerated AKR lymphomagenesis. First, lymphoma cells arise which are initially thymus-dependent and can appear in one or simultaneously in both thymic lobes. These progress to become thymus-independent, fully autonomous, tumor cells. Thymocytes close to or at the time of the initial transformation event show a marked disorder of differentiation defined by the alterations in the CD4, CD8 surface phenotype distribution.

All AKR mice develop thymic lymphoma between 60 and 90 days of age after neonatal treatment with the oncogenic retrovirus SL 3-3. At 40-50 days of age, in the normal-sized thymus of virus-treated mice, cells appear that produce lymphoma when inoculated intrathymically but not when inoculated s.c. These cells are designated as thymus-dependent (TD) lymphoma cells. TD cells progress to cells that form tumors after both intrathymic and s.c. inoculation; these are designated as thymus-independent (TI) lymphoma cells. In this report, we show that the TD and TI cells can be distinguished as two distinct cell populations. Experiments show that the TD cells reside within the immature CD4- CD8- thymocyte population of the virus-treated mice. In addition, we also show that CD4- CD8- thymocytes from SL 3-3 virus-treated mice do not mature in fetal thymic stromal rudiments. Using three-color flow cytometry to trace maturation of CD4- CD8- thymocytes after intrathymic inoculation into irradiated syngeneic hosts, disregulated thymocyte maturation of this population from virus-treated mice is demonstrated. Thus, altered maturation of and the appearance of TD lymphoma cells in, the most immature population of thymocytes appears to be a first step in a multistep process of thymic lymphomagenesis caused by SL 3-3 virus.

By using an assay system in which small numbers of murine T lymphoma cells are stimulated to grow in serum-free medium, we have continued and expanded our previous studies of an autocrine growth factor that we call leukemia-derived growth factor (LDGF). We show that a T lymphoma cell line of immature phenotype, adapted to growth in serum-free medium, produces and responds to LDGF. LDGF activity is distinct from activities of 10 highly purified or recombinant hematopoietic growth factors including IL-1 and IL-2. However, growth-stimulating activity for the murine lymphoma cells is provided by a partially purified human LDGF.

The role of tumor cell membrane gangliosides in tumor formation was probed using a series of cloned murine AKR lymphoma cell lines. Tumor formation was directly related to high expression and shedding of membrane gangliosides. In vivo, as little as 1 pmol of purified total gangliosides of highly tumorigenic cells, injected intradermally with poorly tumorigenic cells (which lacked and did not shed gangliosides), markedly increased the tumorigenicity of these cells in syngeneic normal mice. Thus, gangliosides shed by tumor cells are a previously unrecognized, extremely potent enhancer of tumor formation in vivo.

The progression of prelymphoma cells (PLC) in the bone marrow to lymphoma cells (LC) in the thymus in the presence of the lymphomagenic retrovirus SL3-3c was studied in a model system of virus-accelerated thymic lymphoma in AKR/J mice. On the average, a single LC appears in the thymus 30 days after the neonatal ip inoculation of SL3-3c virus; 50 days later, thymic lymphoma is clinically detectable. PLC in the bone marrow and oncogenic virus in the thymus are continuously present during this period before lymphoma develops. Biologically active oncogenic virus in the thymus increases as the animal nears the time of lymphoma development. Intrathymic inoculation, but not ip inoculation, of SL3-3c virus results in accelerated thymic lymphoma in 4- to 6-week-old AKR mice. PLC defined as a population of bone marrow-derived thymocyte progenitor cells susceptible to malignant transformation by oncogenic retrovirus after homing to thymus were further studied and characterized. PLC, like normal bone marrow thymocyte progenitors, were found to be radiosensitive and glucocorticoid resistant. Thymocytes of 21- to 28-day-old AKR mice, neonatally inoculated with SL3-3c virus, were studied for PLC. They could not be detected. It is concluded that lymphoma development is the final outcome of a series of events in which bone marrow-derived thymocyte progenitors are transformed after entering the thymus by virus in the thymic environment.

It has been observed that subclones from the spontaneous murine AKR/J T-lymphoma cell line SL12 with similar in vitro growth characteristics exhibit stable differences in tumorigenicity. The cell line is composed of at least three distinct cloned cell types that are highly, moderately, or poorly tumorigenic in syngeneic host animals. When healthy, young, syngeneic host animals were given iv injections with the same number of viable growth phase cells, each cloned cell type had a different tumor incidence, latent period, and pattern of tumor spread. The unusual stability of the cloned cell lines is shown by a similar incidence, latency, and spread of the tumors when studied after more than 1 year of continuous in vitro culture. The SL12 clones also differ in several phenotypic characteristics commonly used to classify thymocyte maturation, e.g., a) the expression of three of seven surface antigens examined, b) the cellular response to glucocorticoid hormone, and c) the expression of terminal deoxynucleotidyl transferase.

Transferrin has been considered to be an essential requirement for hematopoietic cell proliferation in culture. We have isolated two cloned lymphoma cell lines, SL 12.1 and SL 12.4, which grow and adapt in serum-free medium without added transferrin. Antibody to the transferrin receptor blocks the growth of these cells. We have also demonstrated that transferrin-free conditioned medium from the cells will compete with transferrin for binding. Furthermore, conditioned medium from SL 12.1 and SL 12.4 cells induces and supports exponential growth of a transferrin-dependent lymphoma cell line, SL 12. We conclude that these two transferrin-independent cloned lines produce transferrin-like activity which plays a crucial role for cell proliferation.

Several well characterized murine T-lymphoma cell lines were used in somatic cell hybridization experiments to study the genetic regulation of glucocorticoid-induced lysis. Cell fusions were carried out among the SL12-derived cloned lines and between the W7 and SAK8 lines all of which have functional hormone receptors. These cell lines differ in their sensitivity to glucocorticoid-induced lysis. The resultant hybrids were characterized by their growth response to 1 microM dexamethasone, their hormone receptor content, their chromosome number, and the expression of surface antigens. Fusion of the hormone-sensitive W7 parent to a number of glucocorticoid-resistant cell lines resulted in hybrids which were of the sensitive phenotype. In contrast the fusion of another hormone-sensitive clone, SL12.4, with glucocorticoid-resistant SL12 clones or with SAK8 always resulted in hybrids resistant to glucocorticoid lysis. These results reveal a complex genetic regulation of the hormone response or the requirement for multiple gene activity in the mechanism for glucocorticoid-induced cell lysis.

This study demonstrated the importance of the methods used in determining the lymphoma cell colony stimulating activity of factors derived from lymphoma cells. The in vitro colony formation in a semisolid matrix of the AKR mouse lymphoma cell line, SL 12, and three cloned derivatives, SL 12.1, SL 12.3, and SL 12.4, was studied. We show that the use of soft agar or methylcellulose as a semisolid matrix results in colony formation by the lymphoma cells only in the presence of serum. The addition of conditioned medium (CM) from lymphoma cells growing in serum-free medium does not stimulate colony growth. However, when purified agarose is used, colonies grow in a dose-dependent manner in the absence of serum and in the presence of CM. These results indicate that the type of semisolid matrix used can influence results in studies of this nature. Purified agarose provides the best environment when colony formation by lymphoma cells is used to measure the presence of growth factors in test-conditioned media.

Marrow hypoplasia is described in CBA/H mice that drank water containing 300 mg/liter cadmium chloride for 12 months. This was characterized by a significant reduction of the totipotent stem cells (CFU-s), granulocyte-monocyte progenitor cells (GM-CFUc), and erythroid progenitor cells (CFU-e). The bone marrow cellularity and the proliferative capacity of GM-CFUc in vitro were decreased. The animals reflected these marrow alterations by demonstrating an anemia with reticulocytopenia and neutropenia. They did not show increased mortality or increased susceptibility to infections; however, their body weight was significantly reduced. In addition, iron deficiency was demonstrated in the cadmium-treated mice. The animals had a hypochromia of the peripheral red cells and diminished marrow iron stores. Thus, the anemia of cadmium toxicity is probably the combined result of bone marrow hypoplasia and iron deficiency.