CUBIC: a three-dimensional colored projection of Consort 30 generated trivariate flow cytometric data.

The CUBIC program displays three-dimensional colored dot plots of flow cytometric trivariate data collected by unmodified commercial instruments (FACScan flow cytometer, FACS 440 cell sorter). Assuming a bimodal distribution of the fluorescence intensity of the cells, the eight theoretical subpopulations involved in a three-color fluorescence histogram are clearly localized in the 3-D space by colored dots that are clustered near each corner of a cubic frame. Rotation, tilting, and zoom functions are available. Table look-up is not needed. CUBIC was illustrated by two experiments: 1) three-color immunofluorescence of antigens on human lymphocytes using monoclonal antibodies conjugated either to fluorescein (FITC), to R-phycoerythrin (PE), or to biotin revealed by a streptavidin coupled to a PE-Texas red tandem conjugate (TC); 2) two-color immunofluorescence of CD4 and CD8 antigens on thymocytes of healthy or preleukemic mice correlated to the DNA content quantified by 7-amino-actinomycin D (7-AAD). The three fluorescences were excited by a single argon-ion laser emitting at 488 nm.

Abnormal thymocyte subpopulations in split dose irradiated C57BL/Ka mice before the onset of lymphomas. Effects of bone marrow grafting.

Fractionated whole-body X irradiation (4 x 1.75 Gy at weekly intervals) induces a high percentage of thymic lymphomas in C57BL/Ka mice. The present work reports the phenotypic alterations of thymocyte subpopulations during the preleukemic period: there were a decrease of CD4+ CD8+ cells and an increase of CD4- CD8- and CD4- CD8+ cells. Marrow grafting early after irradiation that prevents lymphoma development restores the thymocyte subpopulations. In many instances, transplantation of 'preleukemic thymocyte inoculate' gives rise to an active and long lasting repopulation of recipient thymuses. However in all cases, donor lymphomas can develop after inoculation of 'preleukemic thymocyte inoculate'.

Cellular events in radiation-induced lymphomagenesis.

Fractionated whole-body irradiation induces thymic lymphomas in most of treated C57Bl/Ka mice. The cellular events occurring during the latency period consist of the emergence of preleukaemic cells and of marked alterations to the T-cell lineage and the microenvironment within the thymus. The proportions of the various thymocyte subsets are modified, suggesting a blockage in the normal differentiation process. Thymic epithelial cells are functionally modified, leading to decreased interactions with immature thymocytes. Interestingly, bone marrow grafting early after irradiation, which inhibits the development of lymphomas, induces the disappearance of preleukaemic cells from the thymus, whereas thymocyte subpopulations and thymic epithelium are restored. Interferon gamma and tumor necrosis factor alpha also prevent the onset of lymphomas. Studies on the effect of bone marrow transplantation and cytokine inoculation in split-dose irradiated mice should allow characterization of the factors that modulate the progression of preleukaemic cells towards the neoplastic state.

Effect of interferon-gamma and tumor necrosis factor-alpha on lymphoepithelial interactions within thymic nurse cells.

Isolated thymic nurse cells (TNC) represent a specialized microenvironment in vivo where thymocytes interact specifically with subcapsular epithelial cells. They are thought to play a critical role in the process of T cell differentiation. We demonstrate that recombinant murine interferon-gamma and recombinant human tumor necrosis factor-alpha can act on these interactions: they stimulate TNC-derived epithelial cells to establish interactions with thymocytes in vitro and to form new lymphoepithelial complexes. This phenomenon is partially inhibited by anti-Ia monoclonal antibodies. Implications of these findings for normal intrathymic differentiation are discussed.

Cellular aspects of the pathogenesis of radiation--induced thymic lymphomas in C57 BL mice (review).

Radiation-induced thymic lymphomas in C57Bl/Ka mice are interesting models for studying the successive steps of carcinogenesis. Irradiation initiates "preleukemic" cells, which are promoted to become neoplastic. Studies in mice in which lymphoma development is inhibited by a bone marrow transplantation after irradiation suggest that radiation--induced alterations to the T cell lineage, and particularly to thymic microenvironment, are critical for the promotion of preleukemic cells. It is proposed that the lack of physiological differentiation signals within the thymus, as a result of irradiation, allows these cells to escape the normal controls of thymocyte production and pushes them towards neoplastic transformation. A disturbance in the production of cytokines may be involved, since exogenous cytokines, such as Interferon gamma or Tumor Necrosis Factor a, can inhibit radiation-induced lymphomagenesis, reproducing the effects of bone marrow transplantation. The model is thus suitable for studying the mechanisms of carcinogenesis and designing biological manipulation devoted to cancer prevention in individuals who have been exposed to oncogenic agents.

Further studies on the mechanism of radiation induced thymic lymphoma prevention by bone marrow transplantation in C57BL mice.

Whole body fractionated irradiation induces thymic lymphomas in C57BL/Ka mice after a latent period during which intrathymic lymphopoiesis is modified; thymocyte numbers are subnormal and the epithelial component of thymic nurse cells (TNCs) is altered as estimated by the number of TNCs in vivo and by its ability to interact with immature thymocytes in vitro. A graft of normal bone marrow cells immediately after the last irradiation prevents the development of lymphomas; but when such a graft is performed 1 month later, it does not inhibit the emergence of tumors. In both cases the grafted precursors home and repopulate the thymus. However, the delayed graft does not exert any effect upon the altered epithelial component of TNCs, whereas the early one restores the numbers of TNCs and the function of their epithelial component. The results thus demonstrate that lymphoid thymic repopulation by a bone marrow graft is not sufficient to prevent the development of lymphomas and that there is an intimate relationship between tumor development and alterations of nurse cells microenvironment.

Control of lymphoepithelial interactions within thymic nurse cells by gamma-interferon and tumor necrosis factor alpha. Possible role in the modulation of intrathymic education?

The role of cytokines in the process of intrathymic differentiation is not yet clearly established. The results presented in this paper demonstrate that interferon-gamma and tumor necrosis factor-alpha regulate 'in vivo' and 'in vitro' the interactions between epithelial cells and thymocytes within thymic nurse cells. The mechanism of action of these cytokines and their possible physiologic role 'in vivo' are discussed.

[Cytofluorimetric study of ploidy in the preleukemic thymus in C57BL/Ka mice under fractionated irradiation]. / Etude cytofluorimétrique de la ploïdie dans le thymus préleucémique chez la Souris C57B1/Ka soumise à une irradiation fractionnée.

The ploidy of the thymus was studied in C57B1/Ka mice irradiated with 4 weekly X-Ray doses of 1.75 Gy. The determination of nuclear DNA content was performed by flow cytometry of intact thymocytes labeled with propidium iodide in presence of a mixture of chicken and rainbow trout red blood cells as internal reference standards. The method has been tested by detecting the sex difference in DNA content of G0/G1 of normal thymic mouse cells. The mean value was 2.9% higher in female mice. The thymus of almost 60% of irradiated male mice present a slight hypoploidy of 2.6% one month after the last irradiation.

[Migration of bone marrow cells toward the thymus in C57BL/Ka under fractionated irradiation]. / Migration des cellules médullaires vers le thymus chez les souris C57BL/Ka soumises à une irradiation fractionnée.

Fractionated whole body X-irradiation (4 X 1.75 Gy at weekly intervals) induces a high percentage of thymic lymphomas in C57BL/Ka mice. These tumors develop after a long latency period during which the thymic lymphopoiesis is deeply altered. In the present work, we test wether those modifications are due to lack of prothymocyte homing to preleukemic thymuses. Our results show that the preleukemic state of the thymus don't prevent the homing of normal marrow precursors grafted immediately after an irradiation of 4 Gy. Thus the alterations of thymic lymphopoiesis observed after a leukemogenic irradiation are not due to a modification in the thymus receptivity to thymocyte precursors.

[Interaction of radiation-induced preleukemia cells and bone marrow grafts in C57BL/Ka mice]. / Interactions de cellules préleucémiques radio-induites et de greffe de moelle chez la Souris C57BL/Ka.

The repopulation of thymus inoculated with radiation-induced preleukemia cells was studied in 400 R irradiated mice grafted with normal bone marrow cells. These marrow cells gave rise to an actively regenerating thymic progeny, as well as in 400 R treated mice receiving only a bone marrow graft. Moreover, the marrow graft did not prevent the progression of inoculated preleukemic cells towards lymphoma growth.

[Relations between the capacity of thymic repeopling and the formation of lymphoma obtained by radiation-induced preleukemic cells in C57BL/Ka mice]. / Relations entre la capacité de repeuplement thymique et de formation de lymphomes exprimée par des cellules préleucémiques radio-induites chez la Souris C57BL/Ka.

We have analyzed the repopulation of thymuses injected with preleukemic cells obtained at various interval after fractionated irradiation. Our results show that preleukemic cells can repopulate the thymus transiently as normal thymocytes or they can proliferate in recipients. In all cases, preleukemic cells can give rise to donor lymphomas.

Phenotype of thymic lymphomas in the mouse.

The MP2 cell line was established from a murine leukemia virus-induced thymic lymphoma. Half of the cells were consistently L3T4 positive and less than 5% of the cells were Lyt-2 positive. Single cell cloning on the basis of the presence or absence of Lyt-2 allowed the isolation of four clones with stable phenotypes: (1) Lyt-2-, L3T4-; (2) Lyt-2+, L3T4+; (3) Lyt-2-, L3T4+; (4) Lyt2+, L3T4-. These data are discussed in relation to tumour cell heterogeneity and to normal T-cell differentiation pathways.

Cellular aspects of radiation leukemogenesis in C57 BL/Ka mice: alterations to thymic microenvironment and lymphopoiesis.

After a leukemogenic split dose course of irradiation, thymic nurse cells (TNCs) disappear. We have correlated this with the loss of an epithelial cell surface antigen (recognized by monoclonal antibody ER-TR3 and tentatively identified as Ia). In addition, epithelial cells have lost their capacity to interact with fetal thymocytes in vitro. Marrow grafting early after irradiation, that prevents the development of lymphomas, restores thymic nurse cells and thymocyte population. Such reconstitution and lymphoma prevention were not observed when marrow grafting was performed later (1 month after irradiation) during the preleukemic period.

[Phenotype of thymocyte populations in C57BL/Ka mice during the development of radiation-induced lymphomas]. / Phénotype des populations thymocytaires chez la souris C57BL/Ka au cours du développement des lymphomes radio-induits.

In C57BL/Ka mice, irradiated with 4 weekly X-ray doses of 1,75 Gy, the phenotype of thymocyte populations was analyzed with monoclonal antibodies, lectins and flow cytometry. Four successive phases were defined: a) early after irradiation, a depletion of radiosensitive cortical thymocytes with a relative enrichment for radioresistant medullary type cells; b) a regeneration phase, during which thymocytes express the cortical immature phenotype; c) a third period, with a relative increase of a population with medullary phenotype; d) a late phase, during which thymocytes with the same phenotype as lymphoma cells accumulate.