Evidence of recombinant ecotropic provirus integration in thymic lymphomas induced by direct or indirect radiation effects.

Several investigators described the occurrence of ecotropic recombinant proviruses in the DNA of in-vivo or in-vitro propagated radio-induced lymphomas, but such proviruses were never detected in primary tumors. To assess their biological significance in the tumorigenic process, we reinvestigated the presence of new proviruses chiefly in primary radio-induced tumors and in models of radioleukemogenesis which could give additional support for their role. Such models included thymic lymphomas originating after (i) graft of non-irradiated thymuses in thymectomized irradiated mice and (ii) the injection of a B-ecotropic retrovirus (T1223/B) in association with a subleukemogenic dose of irradiation. We report for the first time that new ecotropic proviral sequences are encountered in a significant number (30%) of primary lymphomas induced directly by irradiation or indirectly in non-irradiated thymuses grafted in irradiated hosts. The existence of a 3.5-kbp Kpn1 restriction fragment with ecotropic sequences in the digested DNA of these tumor cells indicates that these new sequences belong to an ecotropic provirus recombinant in the gag-pol region. We observed that most of the primary radio-induced tumors in which novel recombinant provirus could be detected, displayed the integration at a single or at a few sites, demonstrating their clonality with respect to viral integration. The same was observed in thymic lymphomas arising after T1223/B virus injection and irradiation and in in-vivo or in-vitro propagated tumors. Altogether, these data bring the first evidence of the integration of ecotropic recombinant proviral genomes in a significant number of primary radiation induced thymic lymphomas and of their possible role in view of their frequent occurrence in grafted thymomas.

Distribution of Lyt antigens on the surface of thymocytes associated with thymic macrophages and dendritic cells.

Thymocyte subpopulations that are associated with macrophages and dendritic cells of the thymus in vivo were isolated from the thymuses of C57B1/6 mice, and their Lyt phenotypes were analyzed. Electron-microscopic examination of immunogold-labeled cells revealed that the thymic complexes formed by macrophages mainly contained Lyt-2-positive thymocytes, while Lyt-1-positive thymocytes were more frequently associated with dendritic cells. The characteristic distributions of Lyt antigens on the surface of thymocytes in regions of reciprocal contact with macrophages (Lyt-2-positive cells) and dendritic cells (Lyt-1-positive cells) suggest that these antigens play a role in specific interactions between thymocytes and stroma cells.

Multicellular complexes of thymocytes and different types of thymic stromal cells in the mouse.

The isolation of multicellular complexes of thymocytes and different types of thymic stromal cells from the mouse thymus is described. Isolated complexes were examined by light microscopy. Stromal cells, binding thymocytes at their surface, were identified using electron microscopy, and three types of epithelial cells, macrophages and interdigitating-like cells (IDC-like cells) were distinguished from their morphological characteristics. The epithelial cell types correspond morphologically to epithelial cells present in situ in various thymic regions. The type of thymocyte-contact with epithelial cells, macrophages and IDC-like cells indicated that the formation of multicellular complexes is common.

Target cells and thymus microenvironment in the pathogenesis of thymic lymphomas in C57BL/Ka mice.

In C57BL/Ka mice, the induction of thymic lymphomas either by inoculation of radiation leukemia virus (RadLV) or by a split dose irradiation requires complex cellular events: Target cells are found among the population of thymic subcapsular blast cells, or, alternatively, of marrow or spleen prothymocytes; Progression of target cells to lymphoma growth requires a multi-step process, which occurs only within thymic microenvironment; Target cells are rapidly induced as "preleukemic" cells; After inoculation of RadLV, the initial events occur when target cells are in close association with cells of a specialized component of thymic epithelium, i.e., the so-called "nurse cells"; The leukemogenic agents induce damages to the thymic microenvironment itself; Lymphoma prevention by marrow grafting after irradiation results from mechanisms still unknown which inhibit the progression of "preleukemic" cells to neoplastic growth.

Isolation of follicular dendritic cells from human tonsils and adenoids. I. Procedure and morphological characterization.

Follicular dendritic cells have been isolated from human tonsils and adenoids and characterized at the ultrastructural level. Follicles were dissected and digested with different hydrolytic enzymes. The cells were separated by sedimentation at unit gravity. By this procedure we obtained follicular dendritic cells enveloping lymphocytes with their cytoplasmic extensions in a way analogous to that described for isolated thymic nurse cells. The ultrastructural features of isolated follicular dendritic cells are similar to those observed in situ. Prolonged enzymatic action caused loss of the enveloped lymphocytes.

[Lympho-epithelial interactions in the thymus during development of radio-induced lymphomas in C57BL mice]. / Interactions lympho-épithéliales dans le thymus au cours du développement des lymphomes radio-induits chez la souris C57BL.

In C57BL/Ka mice, leukemogenic fractionated whole-body X-irradiation induces alterations of the lymphoepithelial interactions normally found in the Thymic Nurse Cells (TNCs) and leads to the disappearance of these complexes. This phenomenon is due to the disturbances of thymic lymphopoiesis caused by modifications of bone marrow prothymocytes and of the epithelial component of TNCs.

Thymic nurse cells and thymic repopulation after whole body sublethal irradiation in mice.

Thymic Nurse Cells (TNCs) are lymphoepithelial complexes which are thought to play a role in the early stages of the intrathymic differentiation pathway. Therefore, their repopulation kinetics were analyzed in mice after sublethal whole-body irradiation. Changes of the number of TNCs per thymus were parallel with the evolution of the whole thymocyte population. Particularly, a first wave of TNCs restoration was followed by a secondary depletion and a final recovery. This suggests that TNCs restoration is related to the proliferating progeny of intrathymic radioresistant thymocytes. When normal bone marrow cells were grafted intravenously after irradiation, no secondary depletion was found. This pattern of restoration was obviously related to thymic repopulation by cells which were derived from the inoculated bone marrow. Homing studies with FITC labelled bone marrow cells showed that inoculated bone marrow cells did not penetrate TNCs early after irradiation. Later on, when immigrant cells started to proliferate, they were found preferentially within TNCs before spreading in the whole thymus. The results indicate that interactions between immature thymocytes and epithelial cells within TNCs are critical for the first steps of intrathymic lymphopoiesis.

Correlation of alkaline phosphatase activity to normal T-cell differentiation and to radiation leukemia virus-induced preleukemic cells in the C57BL mouse thymus.

Cytochemical methods at the light and electron microscopic level were used to define the pattern of alkaline phosphatase (APase) activity in normal thymus and to study its modifications after inoculation with the thymotropic leukemogenic radiation leukemia virus in correlation with the emergence of preleukemic cells and their thymus dependency. APase was found in numerous lymphoblasts of the fetal thymus. The enzyme was also detected in a few lymphoid blast cells of the normal young adult thymus, which were closely associated with thymic nurse cells. The observed distribution of APase in normal thymus suggests that its expression could be limited to an early stage of the T-cell differentiation pathway. After inoculation with radiation leukemia virus, APase activity remained normal for almost the entire latency period, during which virus replication spread to the cortex and thymus-dependent preleukemic cells appeared. An important increase in the number of APase-positive cells occurred later, i.e., at the end of the latency period, in nontumoral thymus, which displayed lymphocytic depletion and contained autonomous thymus-independent preleukemic cells. These latter features obviously reflected the malignant transformation of thymus lymphoblasts, which eventually led to the development of the thymic lymphomas. The results raise the question of the possible filiation between the thymic nurse cell-associated APase-positive lymphoid cells of the normal thymus and the target cells susceptible to productive infection and to neoplastic transformation after radiation leukemia virus infection.

Thymic nurse cells account for the thymus dependency of preleukemic cells in mice after inoculation of radiation leukemia virus.

Inoculation of Radiation Leukemia Virus (RadLV) into C57BL/Ka mice induces thymic lymphomas after a 3-6 month latent period. The leukemogenic process requires a sequence of events from the productive infection of susceptible target cells and induction of preleukemic cells to irreversible neoplastic transformation. Preleukemic cells were detected in the thymus during the first week following virus injection. The thymus dependency of these cells was shown to depend transiently upon peculiar lymphoepithelial complexes called "Thymic Nurse Cells" (TNCs). Indeed, the first preleukemic cells appearing in the RadLV-inoculated thymuses were observed selectively within TNCs. They remained closely associated with these complexes during the first 2 or 4 weeks. Later on, TNCs disappeared almost completely whereas non-TNCs associated preleukemic cells were found. Lymphoepithelial interactions within TNCs were thus required for the initial events of RadLV-induced lymphomagenesis. The subsequent TNCs depletion expressed a disturbance of thymic lymphopoiesis in relation with the neoplastic process.

Thymic nurse cells are the first site of virus replication after inoculation of the radiation leukemia virus.

The induction of thymic lymphomas by inoculation of the Radiation Leukemia Virus (RadLV) requires interactions between RadLV, lymphoid cells and thymus microenvironment. The possible localization of this interaction within the peculiar lymphoepithelial complexes called 'thymic nurse cells' (TNCs) has been investigated. Electron microscopic studies, as well as in vitro experiments using a very sensitive infectious centre detection assay demonstrated that most of the first virus producing cells after RadLV inoculation are located within the TNCs. Most of these structures belong to the thymus subcapsular zone. They contain lymphoid cells with the phenotype of the major (cortical) thymocyte population. Data support the view that a limited subpopulation of subcapsular immature thymocyte can act as specific targets for productive infection with RadLV. Furthermore, the initiation of virus replication appears related to the interaction between the immature thymocyte and the 'nurse cells' microenvironment.

[Migration of bone marrow cells to the thymus in mice after a single sublethal-dose irradiation]. / Migration des cellules de moelle hématopoïétique vers le thymus chez la souris après une irradiation unique à dose sublétale.

In sublethally irradiated mice, thymus repopulation is due first to the proliferation of surviving thymocytes followed by the multiplication of bone marrow derived prothymocytes. The migration of bone marrow cells to the thymus after a single sublethal whole-body X irradiation was studied by using fluorescein isothiocyanate as a cell marker. Irradiation increases the permissiveness of the thymus to the immigration of bone marrow cells. Furthermore, the post-Rx regenerating bone marrow cells exhibit migration capacities greater than the normal ones. The radiation induced changes in the bone marrow thymus interaction might play an important role in thymus regeneration after sublethal irradiation.

[Cytochemical detection and cytofluorimetric analysis of alkaline phosphatase in continuous lines of thymic lymphomas induced in mice by a radiation leukemia virus]. / Détection cytochimique et analyse cytofluorimétrique des phosphatases alcalines dans des lignées continues de lymphomes thymiques induits chez la Souris par le virus des radioleucoses.

Alkaline phosphatase was studied in cell lines established from Radiation Leukemia Virus induced thymic lymphomas of C57BL/Ka mice. The cytochemical staining techniques and flow cytofluorimetry analysis described by Dolbeare et al. (J. Histochem. Cytochem., 1980, 28, 419-426) were used. The alkaline phosphatase found in lymphoma cells was heat labile and L-homoarginine. L-phenylalanine and p-bromotetramisole sensitive and is probably similar to the isoenzyme present in mouse placenta, kidney and liver. Very little alkaline phosphatase activity was detected in normal thymus of adult mice, suggesting that the method used in the paper could be helpful for studying the emergence of the first neoplastic cells during the leukemogenic process.

Thymic microenvironment and cultures derived from mouse thymic explants. A morphological study.

Cultures derived from thymus fragments of embryonic (18-19 day old), newborn or one month old C57BL mice have been characterized functionally l(phagocytic and nonspecific esterase activities) and morphologically by means of light, scanning (SEM) and transmission (TEM) electron microscopy. The observations show the heterogeneity of the cell populations composing the monolayers. After a few days incubation macrophages appear as the predominating cell type, while epithelial cells usually constitute no more than 30% of the cells. Experiments designed to determine the fate of lymphocytes adhering to the monolayers lead us to believe (on the basis of SEM morphometric analysis) that the survival of lymphocytes attached either to thymic macrophages or to epithelial cells is improved during the first days of coculture. This survival enhancement does not, however, appear to be a specific inductive effect since a similar survival increase is found when lymphocytes adhere to non-thymic cells. In contrast with the monolayer, the explant provides a three-dimensional culture system able to preserve intact thymic microenvironmental conditions since numerous lymphocytes are found even in five week old cultures which were not overlaid with thymocytes or spleen cells.