Toshihiko Sado

Age-related change in the potential of bone marrow cells to repopulate the thymus and splenic T cells in mice

Bone marrow chimeras were produced between various combinations of young and old mice using either C57BL/6 mice only or a combination of C57BL/6 and B10.Thy-1.1 mice. The wet weight of the thymus and the number of thymocytes and splenic T cells of donor origin were assessed at appropriate intervals after the bone marrow transplantation. It was revealed that the old bone marrow was inferior to young in terms of the capacity to repopulate the thymus and splenic T cells. Moreover, some age-related qualitative changes appeared to occur in the thymocyte progenitors, as the composition of Lyt phenotype of donor-type T cells in the spleen was different between chimeras produced with young bone marrow and those with old.

Immunohistological analysis of immigration of thymocyte-precursors into the thymus: Evidence for immigration of peripheral T cells into the thymic medulla

The immigration route of thymocyte precursors into the thymic microenvironment was examined in various experiments using two strains of mice (B10.Thy-1.1 and C57BL/6) that were identical in H-2 and different in Thy-1 locus. The experiment of thymus grafting revealed that there were two types of thymocyte precursors; one immigrated into the cortex and vigorously proliferated and the other directly immigrated into the medulla. Such a direct immigration of host-type cells into the medulla of the grafted thymus was not observed, when thymus was grafted into young adult nude mice having no T cells. When bone marrow cells were iv injected into intact mice, the direct immigration of donor-type cells was observed only in the cortex, not in the medulla. In parabiotic mice, the immigration of partners cells into the medulla was observed independently before the proliferation of partners cell in the cortex. These findings taken together indicate that peripheral T cells directly immigrate into and recirculate through the thymic medulla.

Spectrum of Znfn1a1 (Ikaros) Inactivation and its Association with Loss of Heterozygosity in Radiogenic T-Cell Lymphomas in Susceptible B6C3F1 Mice

Abstract Kakinuma, S., Nishimura, M., Sasanuma, S-I., Mita, K., Suzuki, G., Katsura, Y., Sado, T. and Shimada, Y. Spectrum of Znfn1a1 (Ikaros) Inactivation and its Association with Loss of Heterozygosity in Radiogenic T-Cell Lymphomas in Susceptible B6C3F1 Mice. Radiat. Res. 157, 331 – 340 (2002). Ikaros (now known as Znfn1a1), a Krüppel-type zinc-finger transcription factor that plays a critical role in both lineage commitment and differentiation of lymphoid cells, has recently been shown to function as a tumor suppressor gene. We have previously reported a high frequency of LOH (∼50 %) at the Znfn1a1 locus in radiation-induced T-cell lymphoma in susceptible B6C3F1 mice. The aim of the present study was to delineate the types of Znfn1a1 inactivation, with special reference to the LOH status, and to determine the relative contribution of each type of Znfn1a1 inactivation in radiation-induced T-cell lymphomas in B6C3F1 mice. We demonstrated that Znfn1a1 was frequently altered (in ∼50 % of T-cell lymphomas), and that its inactivation was caused by a variety of mechanisms, which came under one of the following four categories: (1) null expression (14 %); (2) expression of unusual dominant-negative isoforms (11 %); (3) amino acid substitutions in the N-terminal zinc-finger domain for DNA binding caused by point mutations (22 %); (4) lack of the Znfn1a1 isoform 1 due to the creation of a stop codon by insertion of a dinucleotide in exon 3 (3 %). The null expression, amino acid substitutions, and dinucleotide insertion inactivation types were well correlated with LOH at the Znfn1a1 allele (86 %) and were consistent with Knudsons two-hit theory. On the other hand, T-cell lymphomas expressing dominant-negative Znfn1a1 isoforms retained both alleles. These results indicate that Znfn1a1 inactivation takes place by a variety of mechanisms in radiation-induced murine T-cell lymphomas and is frequently associated with LOH, this association depending on the type of inactivation.

The characterization of the monoclonal antibody Th‐10a, specific for a nuclear protein appearing in the S phase of the cell cycle in normal thymocytes and its unregulated expression in lymphoma cell lines

A monoclonal antibody (Th‐10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG2a and had κ light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea‐treated B10 mice using Th‐10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC‐conjugated anti‐BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th‐10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G1, G2 and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th‐10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th‐10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein.

Graft-versus-leukemia Effect In Mhc-compatible And -incompatible Allogeneic Bone Marrow Transplantation Of Radiation-induced, Leukemia-bearing Mice

Manifestation of graft-versus-leukemia (GVL) effect in MHC-compatible and -incompatible, allogeneic bone marrow transplantation and the roles of T cell subsets contaminated in the donor bone marrow were studied using radiation-induced leukemia-bearing C3H mice maintained under specific-pathogen-free (SPF) condition. The results indicated that BMT from MHC-incompatible allogeneic (BIO) donor significantly improved the survival of the treated mice as compared with that from syngeneic (C3H) donor. When donor (BIO) bone marrow cells were treated with either anti-Thy 1.2 or anti-Lyt 2.2 antibody plus complement prior to BMT, a beneficial GVL effect was completely abolished. On the other hand, BMT from MHC-compatible allogeneic donors (B10.BR, CBA, AKR) failed to show an improvement in survival. However, intentional enhancement of GVH reaction by preimmunization of B10.BR donor mice with a relatively small number (104∼105) of C3H spleen cells or by an addition of B10.BR lymph node cells to the donor bone marrow resulted in a significant improvement in survival. The depletion of all T cells completely abrogated the GVL effect, while the depletion of either Lyt 2+ or L3T4+ T cells from donor (B10.BR) bone marrow resulted in only partial, if any, abrogation of GVL effect. The results indicate that GVL effect observed in leukemic mice treated with allogeneic BMT from MHC-compatible (B10.BR) and -incompatible (BIO) donors was totally dependent on T cells contaminated in the donor bone marrow, and suggest that the roles of T cell subsets in the induction of GVL effect were different between MHC-compatible (B10.BR) and -incompatible (BIO), allogeneic BMT.

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.

Early decline of thymic effect on T cell differentiation

Thymic lobes of B6C3F1 mice ranging in age from 1 day to 11 weeks were implanted under the kidney capsule of T cell deprived syngeneic young adult TXB mice, and the capacity of the thymus grafts to influence the maturation of T cells was assessed at 6 and 12 weeks after the implantation in terms of (a) regenerative activities of the grafted thymus, (2) splenic T cell dependent anti-SRBC response, and (c) mitogenic reactivity of spleen and lymph node cells to T cell specific mitogens. The results revealed that: (1) thymic tissues from 1 week old donors were most efficient in restoring the immune potential of adult TXB mice; (2) a decline in mitogenic reactivities of spleen and lymph node cells was observed in recipients of thymus grafts from donors of 1 month and older; and a decline of splenic helper T cell function was observed in recipients of thymus grafts from 11 weeks old donors. The significance of this early decline in the thymic effect on T cell differentiation is 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.

H-2 restriction specificity of T cells from H-2 incompatible radiation bone marrow chimeras: further evidence for the absence of crucial influence of the host/thymus environment on the generation of H-2 restricted TNP-specific T lymphocyte precursors

Experiments were conducted to answer the questions related to (a) the role played by the antigen-presenting cells (APCs) present within the thymus and (b) the effect of radiation dose to the recipients on the H-2 restriction profile of TNP-specific cytotoxic T lymphocyte precursors (CTLP) recovered from spleens and/or thymuses of H-2 incompatible radiation bone marrow chimeras (BMC). The H-2 restriction profile of intrathymically differentiating TNP-specific CTLPs was also analyzed in order to test an argument that donor-H-2 restricted CTLP detected in spleens of H-2 incompatible BMC were due to the extrathymically differentiated T cells under the influence of donor-derived lymphoreticular cells. The results indicated the following: (i) splenic T cells from B10(H-2b) leads to (B10(H-2b) leads to B10.BR(H-2k)) chimeras, which were constructed by irradiating primary B10 leads to B10.BR chimeras with 1100 R and reconstituting them with donor-type (B10) bone marrow cells as long as 8 months after their construction, manifested restriction specificities for both donor- and host-type H-2, (ii) splenic T cells from two types of (B10 X B10.BR)F1 leads to B10 chimeras which were reconstituted after exposure of the recipients with either 900 or 1100 R with donor-type bone marrow cells generated both donor- and host-H-2 restricted TNP-specific cytotoxic T cells, and (iii) the TNP-specific CTLPs present in the regenerating thymuses of B10.BR leads to B10 and (B10 X B10.BR)F1 leads to B10 chimeras 4 weeks after their construction were also shown to manifest both donor- and host-H-2 restriction specificities. The significance of these findings on the H-2 restriction profile of CTLP generated in BMCs is discussed.