Hitoko Kamisaku

Immediate and Long-term Effects of Radiation on the Immune System of Specific-pathogen-free Mice

Studies on the immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice are summarized in this paper. There was a striking difference in the radiation response of lymphocyte subsets; B cells consist of a fairly radiosensitive homogeneous population, whereas T cells consist of a large percentage (greater than 90 per cent) of radiosensitive and a small percentage (less than 10 per cent) of extremely radioresistant subpopulations. Ly 1+ and Ly 2+ lymphocytes appear equally radiosensitive, although the percentage of radioresistant cells was slightly larger for the former (approximately 5.5 per cent) than the latter (approximately 2.5 per cent). There was a significant strain difference in the radiosensitivity of immune-response potential in mice; immunocompetent cells of C3H mice were more radioresistant than those of BALB/c, C57BL/6, and B10.BR mice. Studies on the long-term effect of radiation on immune system in mice indicated no evidence for accelerated ageing of the immunologic functions when radiation exposure was given to young adults. Preliminary results on the enhancing effect of low dose radiation on cytotoxic T cell response in vitro are also discussed.

Cellular basis of the immunohematologic defects observed in short-term semiallogeneic B6C3F1 → C3H chimeras: Evidence for host-versus-graft reaction initiated by radioresistant T cells

Lethally irradiated C3Hf mice reconstituted with a relatively low dose (2 × 106) of B6C3F1 bone marrow cells (B6C3F1 → C3Hf chimeras) frequently manifest immunohematologic deficiencies during the first month following injection of bone marrow cells. They show slow recovery of antibody-forming potential to sheep red blood cells (SRBC) as compared to that observed in syngeneic (C3Hf → C3Hf or B6C3F1 → B6C3F1) chimeras. They also show a deficiency of B-cell activity as assessed by antibody response to SRBC following further reconstitution with B6C3F1-derived thymus cells 1 week after injection of bone marrow cells. A significant fraction of B6C3F1 → C3Hf chimeras was shown to manifest a sudden loss of cellularity of spleens during the second week following injection of bone marrow cells even though cellularity was restored to the normal level within 1 week. The splenic mononuclear cells recovered from such chimeras almost invariably showed strong cytotoxicity against target cells expressing donor-type specific H-2 antigens (H-2b) when assessed by 51Cr-release assay in vitro. The effector cells responsible for the observed anti-donor specific cytotoxicity were shown to be residual host-derived T cells. These results indicate strongly that residual host T cells could develop anti-donor specific cytotoxicity even after exposure to a supralethal dose (1050 R) of radiation and that the immunohematologic disturbances observed in short-term F1 to parent bone marrow chimeras (B6C3F1 → C3Hf) were due to host-versus-graft reaction (HVGR) initiated by residual host T cells. The implication of these findings on the radiobiological nature of the residual T cells and the persistence of potentially anti-donor reactive T-cell clones in long-surviving allogeneic bone marrow chimeras was discussed.

Limiting dilution analysis of T-cell progenitors in the bone marrow of thymic lymphoma-susceptible B10 and-resistant C3H mice after fractionated whole-body X-irradiation

Earlier studies from this laboratory using Thy 1 congenic B10 strain mice suggested that a depletion of T cell progenitors (pre T cells) in the bone marrow in addition to the destruction of the thymus after fractionated whole body X-irradiation (IR) are the two main critical factors that cause differentiation arrest of initially repopulating intrathymic radio-resistant T cell progenitors, which then lead to the appearance of preneoplastic, prelymphoma cells, and eventually to highly neoplastic thymic lymphomas under the influence of the thymic environment. In order to explore the significance of the depletion (or reduction) of T cell progenitors in the bone marrow during pathogenesis of radiation-induced thymic lymphomas, we compared the pool size of pre T cells in the bone marrow and the spleens as well as the profiles of the regenerating thymocyte populations between thymic lymphoma induction-susceptible B10 and -resistant C3H strain mice following irradiation. The results indicated that irradiation severely depleted the pre T cells in the bone marrow and the spleens of both lymphoma induction-susceptible and -resistant mice. They also showed that in C3H mice the differentiation and maturation of intrathymic T cell progenitors which initially repopulated the depleted thymus seemed to proceed normally in spite of the poor cellularity, while this process was greatly suppressed in B10 mice. These data indicate that a depletion of pre T cells in the bone marrow combined with atrophy of the thymus in the irradiated mice is necessary, but not sufficient for development of thymic lymphoma. Implication of these findings on the possible mechanism of radiation-induced thymic lymphomagenesis is discussed.

Nature of T-cells resident in spleens of thymectomized, lethally irradiated, bone marrow-reconstituted mice

Abstract Adult thymectomized, lethally irradiated, bone marrow-reconstituted (ATxXB) mice that had been weakly primed with SRBC or HRBC between thymectomy and irradiation were shown to retain antigen-specific immunological memories for at least 1–5 months after bone marrow reconstitution. This could be shown by anamnestic antibody response in vivo as well as by proliferative response of the spleen cells to the test antigens in vitro. Spleen cells taken from ATxXB mice showed a reduced but significant proliferative response to nonspecific T-cell mitogens, in particular to Con A, in vitro. Treatment of the donor bone marrow cells used for reconstitution of ATxXB mice with anti-Thy 1.2 sera + C′ did not affect the generation of immunological memories nor the magnitude of the proliferative response of spleen cells to nonspecific T-cell mitogens in vitro, indicating that the cells responsible for such functions were host derived. Finally, the antibody-forming capacity of spleen cells derived from SRBC-primed ATxXB mice to the test antigen in vitro was completely abrogated by exposure to 450 R, whereas the helper function of the same cell suspension remained unaffected even after exposure to 1000 R. Implication of these findings on the nature of T cells resident in spleens of ATxXB mice was discussed.

Different cellular basis for the resistance of C3H and STS strain mice to the development of thymic lymphomas following fractionated whole-body irradiation: analysis using radiation bone marrow chimeras.

Purpose : B10 strain mice are susceptible to the induction of thymic lymphomas by fractionated whole-body X-irradiation (FI), whereas C3H and STS mice are resistant. The nature of the factors responsible for the strain difference in the susceptibility to thymic lymphomagenesis was investigated by using radiation bone marrow chimeras. Methods : Radiation bone marrow chimeras were constructed in the reciprocal donor-host combinations of susceptible and resistant mice with use of Thy 1 markers that allow the genetic origins of thymocytes and thymic lymphomas to be determined. Results : B10.Thy 1.1 →C3H, B10.Thy 1.1 →STS as well as B10.Thy 1.1 →B10 bone marrow chimeras manifested a high incidence of thymic lymphomas after FI-treatment, whereas C3H.Thy 1.1 →B10 and STS →B10.Thy →1.1 as well as C3H.Thy 1.1 →C3H and STS →STS chimeras manifested a low incidence of thymic lymphoma. Furthermore, FI-treatment of (B10.Thy 1.1 + C3H) →B10.Thy 1.1 mixed chimeras resulted in the generation of similar numbers of thymic lymphomas of B10 and C3H origins, whereas FI-treatment of (B10.Thy 1.1+ STS) →B10.Thy 1.1 mixed chimeras preferentially induced thymic lymphomas of B10 origin. Conclusions : (1) genetic factors responsible for the strain-dependent susceptibility and/or resistance to FI-induced lymphomagenesis exert their effects entirely on bone-marrow derived cells, (2) host environments of C3H and STS resistant mice are not inhibitory for the development of thymic lymphomas and (3) the resistance of STS mice to FI-induced thymic lymphomagenesis is an intrinsic property of thymocytes, whereas C3H and B10 thymocytes themselves are similarly susceptible for FI-induced thymic lymphomagenesis.PURPOSE B10 strain mice are susceptible to the induction of thymic lymphomas by fractionated whole-body X-irradiation (FI), whereas C3H and STS mice are resistant. The nature of the factors responsible for the strain difference in the susceptibility to thymic lymphomagenesis was investigated by using radiation bone marrow chimeras. METHODS Radiation bone marrow chimeras were constructed in the reciprocal donor-host combinations of susceptible and resistant mice with use of Thy 1 markers that allow the genetic origins of thymocytes and thymic lymphomas to be determined. RESULTS B10.Thy 1.1-->C3H, B10.Thy 1.1-->STS as well as B10.Thy 1.1-->B10 bone marrow chimeras manifested a high incidence of thymic lymphomas after FI-treatment, whereas C3H.Thy 1.1-->B10 and STS-->B10.Thy 1.1 as well as C3H.Thy 1.1-->C3H and STS-->STS chimeras manifested a low incidence of thymic lymphoma. Furthermore, FI-treatment of (B10.Thy 1.1+C3H)-->B10.Thy 1.1 mixed chimeras resulted in the generation of similar numbers of thymic lymphomas of B10 and C3H origins, whereas FI-treatment of (B10.Thy 1.1+STS)--> B10.Thy 1.1 mixed chimeras preferentially induced thymic lymphomas of B10 origin. CONCLUSIONS (1) genetic factors responsible for the strain-dependent susceptibility and/or resistance to FI-induced lymphomagenesis exert their effects entirely on bone-marrow derived cells, (2) host environments of C3H and STS resistant mice are not inhibitory for the development of thymic lymphomas and (3) the resistance of STS mice to FI-induced thymic lymphomagenesis is an intrinsic property of thymocytes, whereas C3H and B10 thymocytes themselves are similarly susceptible for FI-induced thymic lymphomagenesis.

Protection of retrovirus-induced disease by transplantation of bone marrow cells transduced with MuLV env gene via retrovirus vector

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). We have demonstrated previously that bone marrow chimeras in which hematopoietic cells were replaced with cells expressing Fv-4 resistant (Fv-4r) gene product became refractory to Friend leukemia virus (FLV)-induced leukemogenesis. To induce in vivo resistance against retrovirus-induced diseases by retroviral vector-mediated gene transduction, we introduced Fv-4 env gene into bone marrow cells of FLV-susceptible C3H/He (C3H) mice with retroviral vector (pLSF) derived from murine Friend spleen focus forming virus (SFFV) and the cells were transplanted into lethally irradiated C3H mice. After the bone marrow transplantation, Fv-4r gene product was successfully expressed on erythroid and myeloid cells, while lymphoid cells were only weakly expressing Fv-4r gene product. The C3H mice expressing relatively higher amounts of Fv-4r gene product were rendered resistant to FLV-induced erythroleukemia, while mice expressing lower amounts of the Fv-4r gene product were still susceptible. Effective protection of FLV-induced leukemia in these mice suggested that the Fv-4r gene expression by erythroid cells that were the major target of FLV infection might be critical for resisting FLV-induced leukemia. Thus, gene therapy model by transducing Fv-4r env gene using bone marrow transplantation would provide a useful protection model system of retrovirus-induced diseases.

Cytogenetic and cellular events during radiation-induced thymic lymphomagenesis in the p53 heterozygous (±) B10 mouse

Purpose : Cellular and cytogenetic events in radiation-induced thymic lymphomagenesis were investigated in the p53 heterozygous (±) mouse following a single dose of whole-body irradiation. Materials and methods : The loss of the wild-type p53 allele and microsatellite markers of chromosome 11 in thymic lymphomas that developed in the p53 heterozygous (±) mouse after irradiation, and the stage at which prelymphoma cells appeared were analysed. Results : The p53 heterozygous mouse developed thymic lymphomas in a dose-dependent manner. The loss of the wild-type p53 allele (loss of heterozygosity; LOH) occurred in almost all thymic lymphomas induced in the irradiated p53 heterozygous mouse. Cytogenetic analysis for the mechanism of LOH strongly suggested that the loss of the wild-type p53 gene in the lymphomas was caused by duplication of the disrupted allele through either homologous recombination or non-disjunctional chromosome duplication. The assay for prelymphoma cells suggested that a critical event in the development of prelymphoma cells occurred at least 3 weeks after irradiation. Conclusions : The loss of the wild-type p53 gene in thymocytes of the p53 heterozygous mouse may precede the development of prelymphoma cells after irradiation and be a valuable marker of radiation-induced leukemogenesis.

Contrasting feature in the repopulation of host-type T cells in the spleens of F1----P and P----F1 radiation bone marrow chimeras.

The regeneration and persistence of host- and donor-derived T cells were examined in the thymus as well as the spleen of mouse radiation bone marrow chimeras of two semiallogeneic combinations (F1----P, P----F1) with different Thy-1 markers on T cells of donor and host origins. An unexpectedly large number of host-type T cells were recovered from the spleens of F1----P chimeras, amounting to as high as 45 and 25% of total T cells at 6 and 14 weeks after bone marrow transplantation (BMT), respectively. To the contrary, the residual host-type T cells in the spleens of P----F1 chimeras disappeared quickly, resulting in less than 0.1% of total T cells at 6 weeks after BMT. It was also revealed that the number of host-type T cells in the spleens of F1----P chimeras decreased in proportion to increase of radiation dose given to the recipients.

Possible induction of graft-versus-leukemia effect against a leukemia refractory to antileukemia response in ordinary MHC-compatible, allogeneic bone marrow transplantation.

We previously indicated that intensity of the graft-versus-leukemia (GVL) effect varied among different leukemias in MHC-compatible, allogeneic bone marrow transplantation (BMT). Cellular factors responsible for differences in intensity of the GVL effect were examined by using two types of leukemias, i.e., a resistant leukemia (LE750) and a sensitive leukemia (8313) to induction of the GVL effect in MHC-compatible, allogeneic BMT of leukemia-bearing host. Resistance of LE750 leukemic cells to induction of the GVL effect could not be attributed to either less sensitivity to lysis by minor H antigen-specific, cytotoxic T cells or to an immunosuppressive activity of LE750 leukemic cells in leukemia-bearing host, when compared with the case of the sensitive leukemia (8313). To investigate the significance of the dose effect of effector cells for induction of the GVL effect, we used CD8+ T cells of AKR donor mice, which were shown to preferentially induce the GVL effect with hardly any lethal graft-versus host disease against C3H recipient mice, enabling us to increase the number of CD8+ T cells used in the allogeneic donor inoculum. The results suggested that the outcome of the antileukemic response in allogeneic BMT of leukemic recipients may be determined, at least in part, by the balance between the size of leukemic cells surviving and repopulating in the recipients after BMT and the number of antileukemic effector cells. The results furthermore indicated that when donors with T-cell subsets that preferentially induce an antileukemic response with reduced graft-versus-host disease are available, a more effective antileukemic response is inducible even against advanced leukemias.