Effect of interleukins and anti-CD3 monoclonal antibody on the proliferation of thymocytes from irradiated mice.

The responsiveness of thymocytes on day 8 after irradiation to mitogens or anti-CD3 monoclonal antibody was evaluated in the presence of interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6) or phorbol-myristate-acetate (PMA). After irradiation, the thymocytes were poorly responsive to T cell mitogens (Concanavalin A, phytohemagglutinin) and the defect could not be overcome by exogenous IL-2, IL-4, IL-6 or by PMA. In contrast, the combination of the calcium ionophore (A23187) and PMA stimulated thymocyte proliferation to a normal level. The anti-CD3 antibody associated with PMA activated thymocytes above the control values, but this was not observed when anti-CD3 was associated with either IL-2 or IL-4. These results suggest that in the thymic populations present early after irradiation 1) the weak proliferative response to mitogens could be related to a defect at a thymocyte level associated or not to an accessory cell deficiency, 2) the intracellular mechanisms involved in T cell proliferation were not altered, 3) the T cell antigen-receptor/CD3 complex was functional.

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.

Autologous monoclonal antibodies recognize tumour-associated antigens in X-irradiated C57BL/6 mice.

X-irradiation of C57BL/6 mice induces thymic lymphosarcomas which sometimes contain retroviruses which upon injection into normal mice mimic the effect of the irradiation. We examined whether specific antigenicities, viral or cellular, were expressed by tumour cells that could be recognized by antibodies from the irradiated animals. We developed monoclonal antibodies (MAbs) using splenocytes of the diseased animal. The reactivity of such MAbs towards thymoma cell lines established in vitro was investigated by means of an ELISA. At least 10 antibody specificities were detected on the 13 tumours investigated, allowing separation of the MAbs into three classes: those recognizing the autologous tumour, heterologous tumours as well as normal thymic tissue, those specific for the autologous tumour, and those specific for one tumour, but not ones of autologous origin. The last two classes corresponded to specific tumour-associated antigens. Our panel of MAbs defined each tumour by the particular pattern of antigens harboured. It is striking that most of the antigens were present in the normal thymus and that only two tumours had additional antigenicities. Additionally, quantitative variations were observed in the levels of expression of these antigens.

Induction of thymic lymphosarcomas in C57BL/6 mice after inoculation of weakly oncogenic viruses associated with a sub-leukemogenic radiation exposure (1.75 Gy x 2).

B-ecotropic retroviruses arise frequently in old or irradiated C57BL/6 mice as a consequence of a genetic recombination between endogenous eco- and xenotropic retroviruses. They are weakly oncogenic and express a very low tropism for thymic cells. However, their activation by X-rays and the subsequent insertion of new proviral sequences in the cell genome of in vivo- and in vitro-passaged tumors suggest that they might play a role in radioleukemogenesis. To study this possibility, a cloned B-ecotropic virus (1223) was injected into C57BL/6 mice subjected to a subleukemogenenic irradiation which induces only 7% of thymic lymphosarcomas (TL). When it was injected prior to or after irradiation, 1223 induced respectively 31% and 19% of TL. The incidence of TL in the different groups closely correlated with virus expression in hematopoietic tissues during the preleukemic period. Thus, irradiation seems to amplify bone marrow (BM) and thymic cell population(s) which play a decisive role in viral expression. A recombinant provirus (presumably the injected 1223) was detected in the genomic DNA of all tumors tested irrespective of the inductive protocol. BM restoration, which does not inhibit TL produced by highly oncogenic passaged viruses, but prevents the development of TL induced by 4 doses of 1.75 Gy, also provided strong protection in the present experiments. The present data support the hypothesis whereby weakly oncogenic B-ecotropic viruses similar to those activated by radiation might be involved in the development of TL.

Murine thymic lymphomas after infection with a B-ecotropic murine leukemia virus and/or X-irradiation: proviral organization and RNA expression.

The role of retroviruses in murine radioleukemogenesis was reinvestigated using a protocol associating the injection of a non-pathogenic retrovirus (T1223/B virus) and a subleukemogenic dose of X-radiation (2 X 1.75 Gy). Using the Southern blotting technique we studied MuLV proviral organization and RNA expression in thymic lymphomas induced by the combined effect of virus and irradiation or irradiation alone. A recombinant provirus was detected in the chromosomal DNA of every tumor induced by associating virus and radiation whereas it was unconstantly found in radio-induced tumors. In every instance, the provirus was not integrated at a common site. No relationship was observed between viral RNA expression and tumor induction. Trisomy 15 was observed in all metaphases irrespective of the protocol of tumor induction. The G-banding technique revealed an extra-band in several thymic lymphomas induced by irradiation and T1223/B virus injection.

Effect of lymphoma grafts on natural killer cell activity in AKR and C57BL/6 mice.

The natural killer (NK) cell activity of spleen suspensions was measured in AKR and C57BL/6 mice grafted either with isogeneic thymic or nonthymic lymphomas. The transplanted cells originated from lymphoid tumors (B, T, or null) which developed either spontaneously (AKR) or after radiation exposure or after injection of retroviruses (C57BL/6). The NK response was significantly enhanced in AKR and C57BL/6 mice grafted with nonthymic and with some thymic lymphoma lines maintained by in vivo passages. The increase of NK activity which took place during the first 5 days after grafting was concomitant with a hyperplasia of the spleen red pulp. Cells from invaded spleens presented a suppressive effect on NK activity. Most primary AKR thymomas and 4 out of 8 tested thymic lymphomas maintained by in vivo passage in C57BL/6 mice were not inducers. In vitro passaged lymphomas, whether AKR or C57BL/6, displayed variable capacity of stimulation which did not match those of the same in vivo maintained lines. It was found that the capacity of most cultured cells to stimulate NK activity correlated positively with the reverse transcriptase concentration of the corresponding culture media.

Thymic lymphosarcomas obtained by X-rays in association with a weakly leukemogenic virus: cellular studies.

The association in C57BL/6 mice of a subleukemogenic radiation dose (1.75 Gy X 2) which induces 7% of thymic lymphosarcomas (TL) with the injection of a weakly oncogenic B-tropic retrovirus responsible for 5% of TL resulted in a higher incidence of TL (31%) than expected from a simple cumulative effect when the viral injection preceded the irradiations (VX protocol). When virus was injected after irradiation (XV protocol) TL incidence (19%) was not significantly different from that of a cumulative phenomenon. The B-tropic virus used (1223) was isolated from RadLV-Rs extract and cloned. The TL incidence correlates with the presence of virus firstly in the thymus and bone marrow (BM) during the preleukemic period, secondly in the cell lines established in vitro from TL obtained in both protocols. This suggests that B-tropic viruses derepressed by 4 radiation doses of 1.75 Gy might be similarly implicated in the mechanism of radio-induced TL. This hypothesis is further supported by the evidence that BM restoration inhibited leukemogenesis in processes induced either by 4 radiation doses of 1.75 Gy or by the association of 2 radiation doses and viral injection whereas it has no effect on TL induced by highly oncogenic thymotropic viruses. Transplantation of BM cells from animals which had been submitted shortly before to leukemogenic VX protocol failed to induce donor type TL or leukemias in irradiated recipients suggesting that preleukemic cells either are not present or cannot be detected. However a high incidence of recipient TL was observed indicating that viruses were transferred with the grafted cells.

Response to ConA and PHA of host and donor thymic cells in radiation bone marrow chimeras.

The response of thymic cells to ConA and PHA was followed during 49 days in 9 Gy-irradiated C3H mice reconstituted with (C3H X AKR)F1 BM. Thymic suspension were fractionated firstly according to their capacity to bind PNA, and secondly by their Thy-1 surface antigen: Thy-1.1 antiserum was used either to lyse doner-derived cells or to separate them from host cells by panning. From days 14 to 25, when the donor-derived elements expand rapidly, the PNA- fraction remains stable and equal to 6%, suggesting that PNA+ and PNA- cell populations develop independently of each other. On the contrary, the PNA- fraction which initially represents 6% of the surviving cells rises up to 12% after day 20 and remains at this level in the long-lived host population until the end of the experiment (49 days). The response to ConA returns to normal as early as 15 days after X-rays in the PNA- fraction, whereas the response to PHA is still impaired at the end of the experiment. In both cases host cells express a higher level of responsiveness to mitogens than donor cells.

Thymic control in expression of natural killer activity in AKR and C57BL/6 mice.

The natural killer (NK) activity of 2-month old AKR mice was markedly depressed compared with that of C57BL/6 mice of the same age. It decreased with age in C57BL/6 mice, whereas it increased and reached a maximum at 5 months in AKR mice. When the animals developed leukaemia, NK activity completely disappeared. A considerable increase in NK activity was observed in AKR and C57BL/6 mice which had been thymectomized at 30 days of age. This activity remained at a high level for 3 months and slowly decreased afterwards. The NK activity of C57BL/6 mice fell after sublethal irradiation (1.75 Gy X 4) and did not recover within 12 weeks, except in mice restored with bone marrow (BM). When mice were thymectomized prior to radiation exposure, NK activity remained at a normal level and BM restoration resulted in a weak but significant enhancement. NK activity, which was already low in AKR mice, was not significantly modified by sublethal irradiation, whether the animals were previously thymectomized or not. In this strain, BM restoration increased the NK activity after sublethal irradiation, and a higher level was reached in thymectomized animals than in intact ones. Lethally irradiated mice restored with bone marrow displayed a high level of NK activity, which was comparable in both strains and was augmented by thymectomy. Taken together, these results indicate that spleen NK activity is, at least in part, controlled by the thymus. The increased NK activity in thymectomized animals might result from the decline or inactivation of a thymus-dependent suppressive activity. The low NK activity observed in AKR mice seems to be related to a thymus-mediated suppressive effect rather than to a defect of BM in NK precursors.

Protective effect of bone marrow and spleen suspensions on radiation-induced leukemogenesis in C57BL/6 mice.

The present experiments are an attempt to precise the type and localization of the cells involved in the protective effect of hemopoietic suspensions against the radiation-induced thymic lymphosarcoma (TLS) of C57BL/6 mice. Inocula containing variable numbers of BM or spleen CFUs from 60-day-old and 360-day-old donors were tested. According to their origin, the suspensions differed with respect to the CFU replication rate, the CFU ability to differentiate towards the T lineage and the content of the suspensions in thymic precursors. Two levels of inhibition were observed: BM suspensions from 60-day-old donors containing 1,500 CFUs had the best protective effect: 14.5% of TLS; 1,500 CFUs from 360-day-old donors were slightly but not significantly less efficient (28.5%). The second level of inhibition (36-46% of TLS) was obtained with all the following inocula: a) 1,200 and 300 spleen CFUs or 300 and 95 BM CFUs from 60-day-old donors, b) 1,500 spleen CFUs from aged donors. Seventy-six spleen CFUs from 60-day-old donors, 120 BM or 175 spleen CFUs from aged donors had no effect. These results suggest that in addition to the high replication rate of the BM CFUs as compared with spleen CFUs, cells endowed with an optimal protective effect are present in BM suspensions and are either absent or present in very small amount in spleen suspensions. These cells which induce an early repopulation of the thymus might correspond to thymic precursors.

[Use of xenografts for the evaluation of tumor chemotherapy. Application to adjuvant chemotherapy]. / Emploi des xénogreffes pour l'évaluation de la chimiothérapie tumorale. Application à la chimiothérapie adjuvante.

Man-mouse xenografts have been widely used to assess the therapeutic activity of carcinostatic drugs on human malignant cells. Xenotransplantation greatly benefited of the use of athymic nude mice and of thymectomized isogenic radiation-chimaeras. However xenografts are still frequently rejected and their growth rate remains low at least for the initial passages. In spite of these limitations they constitute an interesting material which retain most of the characteristics of the original tumour. With few exceptions these grafts express the same sensitivity to chemotherapy as the original tumour and they may therefore provide valuable information in this respects. Unfortunately the long delay of about 5 months necessary to establish a transplantable line and to stabilize its growth parameters limits the use of xenografts in adjuvant chemotherapy. Thus the information brought by the xenografts may at best help in modifying the on-going treatment.

Bovine lymphosarcoma: processing of bovine leukaemia virus-coded proteins.

This report describes pulse-chase experiments performed with cells infected with tumour-derived bovine leukaemia virus (BLV) and using purified gamma-globulins directed against BLV structural proteins, namely gp51, p24, p15 and p12. A gpr72 was found to be the precursor of the gp51 with a gpr70 intermediate. The p12 was shown to be derived from a pr40 with numerous intermediates (pr35, pr22, pr16 and pr14). The p24 and p15 originate from a pr42. Both the pr42 and pr40 are derived from a common pr70. A P45, a P52 and a P27 were also detected. Because these three proteins were found to accumulate progressively in cells and because they were not observed to be processed, they might play a physiological role in infected cells.

Distribution of terminal deoxynucleotidyl transferase activity between peaks I and II in host and donor thymic cells of bone-marrow-restored mice after X-irradiation.

The terminal deoxynucleotidyl transferase (TdT) activity and its distribution between peaks I and II after chromatographic elution were studied on days 15 and 17 after X-irradiation, in host- and donor-derived thymic cells of lethally irradiated (9 Gy) mice restored with BM cells. It was found that the population derived from the surviving host thymocytes differed markedly from the donor-derived population. The cells of host origin has a low TdT activity especially in peak II and the ratio peak I/peak II remained close to 1 instead of 0.1 in controls. These alterations reflect a reduced replication rate and possibly a modification of the cellular metabolic activity (phosphorylation-dephosphorylation). In contrast, the donor-derived elements displayed a very high TdT activity related to their elevated rate of replication, and the ratio peak I/peak II which was close to 1 on day 15 returned rapidly to normal. The impaired replication ability and the metabolic alteration of the host cells might be attributed either to a specific property of the radiation-resistant thymocytes or to residual cellular injury or to a combination of both.

Bovine lymphosarcoma: expression of BLV-related proteins in cultured cells.

Bovine leukaemia virus (BLV) is known to be the aetiological agent of enzootic bovine lymphosarcoma. As the mechanism of tumour induction is unknown, we analysed the viral proteins expressed in cultured bovine cells of different origin, i.e. from enzootic or sporadic tumourous tissues, normal cells infected or not with BLV, and the reference FLK-BLV cells. We also investigated BLVs of different origins. As well as the previously described BLV polyproteins in FLK-BLV cells (pr70 and pr45) we have also found two additional polyproteins, p52 and p27. In these four proteins we observed the combined antigenicities of p24, p15 and p12. We observed an additional polyprotein in p42, with the antigenicities of the p24 and the p15 in cells derived from tumour tissue or infected in vitro with naturally occurring BLV isolates. None of these BLV-coded proteins, nor others with cross-reacting antigenicities, were encountered in non-BLV-producing cultured cells from enzootic or sporadic tumours. The use of autologous sera did not permit the detection of any proteins in addition to the above gag-coded and env-coded proteins. Thus, there is no evidence for the existence of a gag-x fusion protein which could play a role in BLV-induced tumourigenicity.

Terminal deoxynucleotidyl transferase activity in the regenerating thymus of x-irradiated mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) enzyme activity (EU per 10 8 cells) between peaks I and II was followed for a period of 42 days in regenerating thymus of lethally irradiated (9 Gy) C3H mice restored with 10 6 (C3H x AKR) F1 bone marrow cells. The detection of Thy-1.1 and Thy-1.2 surface antigens allowed for the discrimination between host and donor cells, and the main subpopulations of thymic cells were characterized by their sensitivity to H-2 k antiserum and to dexamethazone. Two peaks of TdT activity could be detected on phosphocellulose chromatographic separation. The distribution of TdT activity between these two peaks was followed during the two periods of thymic endo- and exoregeneration. Peak I TdT activity was closely correlated with the variation in the percentage of high H-2 population. Peak II activity was mostly related to low H-2 cells. The per cell content of both peak I and peak II activities exceeded the norm in rapidly expanding populations. Finally between days 10 and 14 the TdT activity of the endoregenerating population was apparently not different from that of the exoregenerating population between days 14 and 22.

Mechanism of early and late polykaryocytosis induced by the bovine leukaemia virus.

Syncytia formation induced by the bovine leukaemia virus (BLV) has been classified as 'early' or 'late' polykaryocytosis. Early polykaryocytosis arises in the first 24 h in mixed cultures of BLV-infected cells with indicator cells. Late polykaryocytosis is observed 4 to 8 days after infection of sensitive cells with cell-free infectious BLV. Our results demonstrate that the two phenomena proceed from different mechanisms. Late polykaryocytosis results from an active process dependent on the integrity of the virus genome. In contrast, early polykaryocytosis is a passive process which does not require any de novo virus synthesis but is dependent on the presence of virus proteins in inducer cells.

Influence of serum thymic factor (FTS) on radiation-induced leukaemogenesis in thymectomized AKR mice.

The influence of serum thymic factor (FTS) on extrathymic leukaemogenesis induced in thymectomized AKR mice by fractionated sub-lethal irradiation was studied. Thirty-day-old thymectomized mice were submitted to four doses at weekly intervals (1.75 Gy) and thereafter treated with FTS (1 ng) for 9 days. Two groups were restored with either bone marrow or spleen cells prior to FTS treatment. In another group mice were treated with FTS (12 ng) for 1 month after thymectomy and prior to irradiation. Results indicated that primarily irradiation and FTS, and, to a lesser extent, restoration and age of the mice at the time of their first radiation exposure influenced leukaemogenesis. Irradiation increased the spontaneous low incidence of extrathymic leukaemia (5%) up to 50%. Although nothing is yet known about the expression of endogenous retroviruses in thymectomized AKR mice, the possible expression of leukaemogenic recombinants, either identical to or different from mink-cell-focus-inducing viruses (MCF), might explain this enhancing effect of radiation on leukaemogenesis. Mice developed two types of leukaemias: "null" leukaemias whose cells bore no detectable theta antigen or surface immunoglobulin, and B leukaemias. Among the null leukaemias, two kinds could be distinguished: "early" ones which were observed before 450 days, and in which BM and spleen were similarly involved, and "late" ones which displayed the same characteristics as B leukaemias, i.e. delayed appearance (after 450 days) and splenic origin. Although FTS did not modify the overall frequency of leukaemias, it increased significantly the incidence of the "early" null ones. It had no effect on the frequency or the latency of late null and B leukaemias. Any population of theta-negative T cells sensitive to FTS could be an acceptable candidate for "early" null leukaemogenesis. The origin of "late" null leukaemias remains an open question.

In vitro production and titration assays of B-tropic retroviruses isolated from C57BL mouse tumors induced by radiation leukemia virus (RadLV-Rs): effect of dexamethasone.

The effect of dexamethasone (DXM) on the replication and titration assays of B-ecotropic radiation leukemia virus (RadLV-Rs) isolates was examined. The drug was shown to enhance in vitro virus release by otherwise low producer permissive cells. DXM also stimulated infectivity and focus formation in murine S+L--permissive cells and shortened the time-appearance of virus-induced foci. In contrast to foci observed in nontreated cultures, which are generally abortive and composed of only a few morphologically transformed cells, those obtained with DXM were comparable to those induced by ecotropic retroviruses with a potent helper activity for murine sarcoma virus. In addition, DXM increased virus-induced XC cell fusion. These fundamental data allowed a more abundant in vitro production of the B-ecotropic RadLV-Rs virus isolates as well as precise infectivity titration assays.