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.

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.

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.

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.

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.

Distribution of Fv-4 resistant gene product in Friend leukemia virus-resistant Fv-4r mouse strain.

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). We demonstrated previously that the Fv-4 resistant (Fv-4r) gene product, Fv-4r env antigen, is released from Fv-4r-bearing BALB/c-Fv-4Wr (C4W) mouse-derived cells into serum in vivo and binds to cells expressing surface receptors for ecotropic MuLV, thereby protecting them from infection with Friend leukemia virus (FLV) by receptor interference. This unique resistance mechanism against retroviral infection might provide a possible therapeutic model system of human retroviral infection such as AIDS. To further investigate the Fv-4r gene action in vivo, we examined the distribution and character of Fv-4r env antigen in serum and systemic organs from C4W mice. The Fv-4r env antigen was immunohistochemically localized to the lympho-hematopoietic cells and exocrine glandular cells, such as those of the salivary gland and pancreas. Using immunoprecipitation followed by Western blotting, we determined two types of gp70-related Fv-4r env antigen in the serum of C4W mice, showing molecular weights of either 70-75 kDa and 80-85 kDa. When thymocytes from Fv-4 susceptible gene (Fv-4r)-bearing C3H mouse were mixed with C4W mouse serum, the 70-75k Da molecule of the C4W serum dominantly bound to C3H thymocytes and thus contributed to receptor interference function. Using immunoelectron microscopy, Fv-4r env antigen was mainly localized to the cell surface membrane of thymic lymphoid cells, while acinar cells of the salivary gland possessed Fv-4r env antigen in the endoplasmic reticulum (ER) as well as on the cell surface membrane. These data indicate that several glandular organs, as well as lymphohematopoietic organs of C4W mice, may contribute to the production of cell-free Fv-4r env antigen, resulting in protection of cells from infection with FLV by receptor interference.

An MHC-compatible allogeneic bone marrow donor with a distinct role of T cell subsets in graft-versus-leukemia effect and lethal graft-versus-host disease.

Our previous results in a murine model indicated that the GVL effect against radiation-induced leukemias could be induced in not only MHC-incompatible but also MHC-compatible allogeneic BMT, and that the intensity of the GVL effect induced in MHC-compatible allogeneic BMT varied among different leukemias and the donor/host strain combinations used. With the use of a radiation-induced T cell leukemia which followed the induction of the GVL effect in both MHC-compatible and -incompatible, allogeneic BMT, the role of T cell subsets in the development of the GVL effect and GVHD was studied. The results indicated that Lyt2+ T cells contaminating donor BM were consistently critical for the induction of the GVL effect in MHC-incompatible (B10) and -compatible (B10.BR and AKR) allogeneic BMT of leukemia-bearing C3H mice, but the depletion of L3T4+ T cells had no effect. In contrast, lethal GVHD induced by AKR donor lymph node cells was totally dependent on L3T4+ T cells, but the depletion of Lyt2+ T cells had no effect. On the other hand, both T cell subsets could cause lethal GVHD induced by MHC-incompatible (B10) and -compatible (B10.BR) allogeneic donors. The distinct roles of T cell subsets of AKR donors were confirmed by the preferential induction of the GVL effect with the AKR donor bone marrow mixed with lymph node cells which had been depleted of L3T4+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-free transmission of Fv-4 resistance gene product controlling Friend leukemia virus-induced leukemogenesis: a unique mechanism for interference with viral infection.

Fv-4 is a mouse gene that dominantly confers resistance to infection by ecotropic murine leukemia virus (MuLV). We previously demonstrated that mixed radiation bone marrow chimeras containing Fv-4r-bearing BALB/c-Fv-4Wr (C4W) bone marrow and Fv-4r-bearing C3H/He (C3H) bone marrow grafted into C3H recipient mice (C4W+C3H-->C3H) were resistant to Friend leukemia virus (FLV)-induced leukemogenesis, even when they contained as high as 70% C3H-derived cells. This indicates that FLV-sensitive C3H-derived cells are rendered refractory to infection and/or transformation with FLV when they coexist in mice with Fv-4r-bearing cells. To investigate the mechanism of Fv-4 resistance to FLV-induced leukemogenesis, we first examined the expression of Fv-4r env antigen in the peripheral blood mononuclear cells (PBMC) of these chimeras. The Fv-4r env antigen was present not only on C4W-derived cells, but also on Fv-4r-bearing C3H-derived cells in C4W+C3H-->C3H mixed bone marrow chimeras. The Fv-4r env antigen that binds to the cells surface of C3H cells was found in sera from normal C4W mice, C4W-->C3H chimeras, and C4W+C3H-->C3H mixed chimeras. The serum Fv-4r env antigen binds to ecotropic MuLV receptors, shown by specific binding to transfectant mink cells expressing ecotropic MuLV receptor, but not to parental mink cells. To determine whether the binding of Fv-4r env antigen to the putative MuLV receptors would block FLV infection, C3H thymocytes or spleen cells that had been preincubated with C4W serum were mixed with FLV and the subsequent production of MuLV specific antigens was examined. C3H thymocytes or spleen cells treated with C4W serum became refractory to binding by FLV. These results provide evidence that the Fv-4r env antigen is released from C4W-derived cells in vivo and binds to cells expressing surface receptors for ecotropic MuLV, thereby protecting them from infection with FLV. The implication of these findings for gene therapy of retrovirus-induced disease such as acquired immune deficiency syndrome (AIDS) is discussed.

Bone marrow transplantation from Fv-4-resistant donors rescues Friend leukemia virus-infected mice from leukemia: a model of bone marrow transplantation therapy against retroviral infection.

Bone marrow transplantation experiments were conducted in mice in order to develop an experimental bone marrow transplantation therapy model, with which to investigate possible means to cure retrovirus-infected hosts with bone marrow or stem cells from virus-resistant donors. In one experiment, lethally irradiated Friend leukemia virus (FLV)-sensitive C3H/He (C3H; Fv-2s, Fv-4s, Rfv-1s, Rfv-2s Rfv-3s) mice were transplanted with (i) bone marrow cells from FLV-resistant BALB/c-Fv-4Wr (C4W; Fv-2s, Fv-4r, Rfv-1s, Rfv-2s, Rfv-3s) mice (C4W --> C3H) or (ii) a mixture of bone marrow cells from C4W and C3H mice (C4W + C3H --> C3H). They were then inoculated with FLV 3-4 months later and leukemia development was examined. The results indicated that C4W --> C3H chimeras were completely resistant to FLV-induced leukemogenesis and that C4W + C3H --> C3H mixed chimeras that contained as low as 30% C4W-derived cells, or as high as 70% C3H-derived cells, did not develop leukemias. In another experiment, bone marrow cells from C57BL/6 (B6; Fv-2r, Fv-4s, Rfv-1r, Rfv-2r, Rfv-3r) or C4W donors were grated to FLV-sensitive DBA/2 mice (DBA; Fv-2s, Fv-4s, Rfv-1s, Rfv-2s, Rfv-3s) that had been infected with FLV 6 days earlier (DBA-FLV). The results indicated that most, if not all, FLV-infected DBA mice which received bone marrow transplantation from B6 donors developed B6-derived leukemias, although the survival time of these mice was dramatically prolonged as compared to that of untreated DBA-FLV mice. In contrast, bone marrow transplantation from Fv-4r-bearing C4W donors successfully rescued FLV-infected DBA mice from leukemic deaths. Thus, the bone marrow transplantation therapy against retroviral infection of hemopoietic cells was shown to be feasible, provided that donor cells carry a resistant allele that functions via receptor blockade as in the case of Fv-4r, but less effective when the roles of the resistant alleles partially interfered with the virus replication, leukemic transformation and/or cycling of target cells as suggested for Fv-2r gene action, or to resist virus infection by immunological means as are known for Rfv-1r, Rfv-2r and Rfv-3r genes which are also carried by B6-strain mice. Implication of these findings on the somatic gene therapy against retrovirus infection diseases in man are discussed.

Graft-versus-leukemia effect in allogeneic bone marrow transplantation in mice against several radiation-induced leukemias.

The intensity of graft-versus-leukemia (GVL) effect was studied using several radiation-induced leukemia cell lines (designated 8027, 8313, 9107, 7929) in MHC-compatible and -incompatible allogeneic bone marrow transplantation (BMT) of leukemia-bearing C3H mice. The results indicated that BMT from MHC-incompatible allogeneic (B10, B10.D2) donors consistently improved the survival of the treated mice compared with that in syngeneic (C3H) donors. However, BMT from MHC-compatible allogeneic (B10.BR, CBA, AKR) donors failed to improve the survival of 8027-bearing recipients. On the other hand, a remarkable improvement in survival of 8313, 9107 or 7929-bearing recipients was observed in MHC-compatible allogeneic (B10.BR, AKR) BMT but there was only a marginal GVL effect in MHC-compatible BMT from CBA donors. There was no clear correlation between the intensity of GVL effect and the amount of class I MHC molecules expressed on leukemic cell lines. The activity of donor lymph node cells for the induction of lethal graft-versus-host disease (GVHD) correlated with the intensity of GVL effect induced by intact donor bone marrow. The results indicate that GVL effect against radiation-induced leukemias could be consistently induced in MHC-incompatible allogeneic BMT whereas the intensity of GVL effect induced in MHC-compatible allogeneic BMT varied among different leukemias and the donor-host strain combinations used.

Friend leukemia virus-induced leukemogenesis in fully H-2 incompatible C57BL/6-->C3H radiation bone marrow chimeras.

Resistance and/or susceptibility for Friend leukemia virus (FLV)-induced leukemogenesis was examined in the fully H-2 incompatible C57BL/6 (B6)-->C3H radiation bone marrow chimeras (RBMC). The results indicated that B6-->C3H chimeras never developed FLV-induced leukemias when infected with FLV 4 months after bone marrow transplantation (BMT). Spleen cells from B6-->C3H chimeras that were preimmunized with 100 Gy-irradiated FBL-3 cells (FLV-induced leukemic cell line originated from B6 mice) were shown to generate anti-FBL-3 specific T-cell proliferation as well as cytotoxic T cells. We also found that when bone marrow cells from B6 mice were mixed with those from C3H mice and then grafted into supralethally irradiated C3H mice, resulting chimeras whose peripheral blood contained less than 30% C3H-derived (susceptible) cells were refractory to FLV-induced leukemogenesis. On the other hand, when C3H mice were infected with FLV and then supralethally irradiated 5 days later and grafted with bone marrow from B6 donors, they developed leukemias which were of B6 origin. Athymic nu/nu mice of B6 background were again shown to develop leukemia following infection with FLV. Possible implication of these findings on the role of T cell-mediated immune response in resistance to FLV-induced leukemogenesis and the immunocompetent nature of fully H-2 incompatible RBMC were discussed.

Bone marrow-thymus interactions during thymic lymphomagenesis induced by fractionated radiation exposure in B10 mice: analysis using bone marrow transplantation between Thy 1 congenic mice.

Bone marrow transplantation (BMT) experiments were conducted using B10.Thy 1 congenic mice to explore the nature of bone marrow-thymus interactions during thymic lymphomagenesis induced by fractionated whole-body X-irradiation (FX). BMT from normal Thy 1 congenic donors into FX-treated recipients one day after FX-treatment resulted in the suppression of tumor development; the suppression being exponentially proportional to the increasing number of bone marrow cells injected. The suppression of tumor development by BMT was shown to be due to prevention of the appearance of prelymphoma cells. BMT from FX-treated donors, which are deficient in pre T cells, into lethally (9 Gy) irradiated Thy 1 congenic recipients resulted in the development of high incidence of thymic lymphomas most of which (approximately 76%) were host-derived, whereas no lymphomas were recovered from the recipients of normal bone marrow. These results suggest that intrathymic T cell precursors which initially repopulate the depleted thymus are prone to undergo preneoplastic changes in the absence of recruitment of more primitive T cell precursors (pre T cells) from the bone marrow but they undergo normal differentiation when large number of pre T cells are available. It was concluded that primary cause of the FX-induced thymic lymphomagenesis was a shortage in supply of pre T cells from the bone marrow to the depleted thymus, which caused differentiation arrest of the progeny of regenerating intrathymic T cell precursors, followed by development of prelymphoma cells that eventually evolve into autonomous lymphoma cells within the thymus.

Phenotypic characterization of thymic prelymphoma cells of B10 mice treated with split-dose irradiation.

Using an intrathymic injection assay on B10 Thy-1 congenic mice, it was demonstrated that thymic prelymphoma cells first developed within the thymuses from 4 to 8 days after split-dose irradiation and were detected in more than 63% of the test donor thymuses when examined at 21 and 31 days after irradiation. Moreover, some mice (25%) at 2 mo after split-dose irradiation had already developed thymic lymphomas in their thymuses. To characterize these thymic prelymphoma cells, the thymocytes from B10 Thy-1.1 mice 1 mo after irradiation were stained with anti-CD4 and anti-CD8 mAb and were sorted into four subpopulations. These fractionated cells were injected into the recipient thymuses to examine which subpopulation contained thymic prelymphoma cells. The results indicated that thymic prelymphoma cells existed mainly in CD4- CD8- and CD4- CD8+ thymocyte subpopulations and also in CD4+ CD8+ subpopulation. T cell lymphomas derived from CD4- CD8- prelymphoma cells had mainly CD4- CD8- or CD4- CD8+ phenotypes. T cell lymphomas developed from CD4- CD8+ prelymphoma cells mainly expressed CD4- CD8+ or CD4+ CD8+ phenotype. T cell lymphomas originating from CD4+ CD8+ prelymphoma cells were mainly CD4+ CD8+ but some CD4- CD8+ or CD4+ CD8- cells were also present. These thymic prelymphoma cells were further characterized phenotypically in relation to their expression of the marker defined by the mAb against J11d marker and TL-2 (thymus-leukemia) Ag, which is not expressed on normal thymocytes of B10.Thy-1.2 or B10.Thy-1.1 strain, but appears on the thymocytes of lymphomagenic irradiated mice. The results indicated that the prelymphoma cells existed in J11d+, TL-2+ cells.

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.

Characterization of virus-specific cytotoxic T cell clones from allogeneic bone marrow chimeras.

We established several H-2-restricted lymphocytic choriomeningitis virus (LCMV)-specific cytotoxic T cell clones from spleens of virus-primed C57BL/6 or C57BL/10 (H-2b) and B10.BR (H-2k) mice and from allogeneic C57BL/10----B10.BR and B10.BR----C57BL/10 bone marrow chimeras. Two T cell clones of H-2b origin and restricted to H-2b, 3 of H-2k origin and restricted to H-2k were compared with two clones each derived from the two types of chimeras. Their surface phenotype was found to be Lyt-2+, L3/T4- and KJ16-133+ (2 of 9). Clones from chimeras expressed bone marrow donor H-2 and are restricted to the recipient H-2. H-2k-restricted clones were all specific for Kk whereas all H-2b-restricted clones were specific for Db. These restriction specificities could be further defined by the blocking activity of various monoclonal anti-H-2 antibodies. Interestingly the anti-H-2Db antibodies blocked the restricted virus-specific killing activity of the clones derived B10.BR----C57BL/10 chimeras much more effectively than the activity of the clones derived from conventional H-2b mice. The various clones differed with respect to their fine specificity for LCMV strains. The 3 clones of conventional B10.BR origin only recognized LCMV-WE but not LCMV-Armstrong, Aggressive or Docile; H-2b-restricted conventional clones recognized target cells infected with all LCMV strains except LCMV-UBC-Docile; the T cell clones from the bone marrow chimeras recognized with one exception all LCMV strains tested.

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.

I-J as an idiotype of the recognition component of antigen-specific suppressor T-cell factor.

The I-J determinant of membrane glycoprotein is known to be expressed exclusively on suppressor T cells (TS), which have a crucial role in the regulation of immune responses. I-J also comprises part of the soluble factor (TSF) with suppressor activity which is secreted from TS. Gene-mapping experiments have indicated that the I-J gene lies between the I-A and I-E subregions of the mouse major histocompatibility complex (MHC) and is defined by the H-2 congeneic pair, that is, B10.A(3R) and B10.A(5R). In fact, antibodies raised in the reciprocal combinations of B10.A(3R) and B10.A(5R) define the I-Jb and I-Jk alleles, and are able to detect the I-J determinants on TS and TSF. Biochemical and functional analyses, using I-J-positive TS clones and hybridomas, have demonstrated that monoclonal anti-I-J antibodies precipitate I-Jk or I-Jb with a relative molecular mass of 25,000-28,000 (25-28K) and that the I-J+ molecule mediates the restriction specificity of TSF in association with an antigen-binding protein (45K). However, molecular genetic studies on the I-J gene reveal no genetic difference between B10.A(3R) and B10.A(5R) and also that there is no room to accommodate a gene encoding I-J in the expected I region. These discrepancies between the molecular genetic and serological/functional data require explanation. Here we demonstrate that TS and TSF expressing I-J of the host type were produced by fully allogeneic bone marrow cells of donor origin in chimaeric mice, when the chimaeras received the host antigen-presenting cells (APC) at the time of immunization. The results show that APC are necessary for the activation and clonal expansion of TS and also support the notion that I-J is an idiotypic determinant of the recognition component of TS and TSF.

Intrathymic T cell differentiation in radiation bone marrow chimeras and its role in T cell emigration to the spleen. An immunohistochemical study.

Immunohistochemical studies were made on the regeneration of T cells of host- and donor-type in the thymus and spleen of radiation bone marrow chimeras by using B10- and B10.BR-Thy-1 congenic mice. Both the thymic cortex and the medulla were first repopulated with thymocytes of irradiated host origin, restoring the normal histologic appearance by days 11 to 14, regardless of the H-2 compatibility between the donor and the host. In Thy-1 congenic chimeras, thymocytes of donor bone marrow origin, less than 100 cells in one thymic lobe, were first recognized at day 7, when the thymus involuted to the smallest size after the irradiation. The thymocytes of donor-type then proliferated exponentially, showing a slightly faster rate when higher doses of bone marrow cells were used for reconstitution, reaching a level of 100 million by day 17 and completely replacing the cortical thymocytes of host origin by day 21. The replacement of cortical thymocytes started from the subcapsular layer in a sporadic manner. The replacement of medullary thymocytes from host- to donor-type occurred gradually between days 21 and 35, after the replacement in the cortex was completed. In the spleen, about 1 million survived cells were recovered at day 3 after the irradiation, and approximately 60% of them were shown to be host-type T cells that were observed in the white pulp areas. The host-type T cells in the spleen increased gradually after day 10, due to the influx of host-type T cells from the regenerating thymus. Thus a pronounced increase of T cells of host-type was immunohistochemically observed in the splenic white pulp between days 21 and 28, when thymocytes of host-type were present mainly in the thymic medulla. These host-type T cells were shown to persist in the spleen for a long time, as long as 420 days after the treatment. Phenotypically, they were predominantly Lyt-1+2+ when examined at day 28, but 5 mo later, they were about 50% Lyt-1+2+ and 50% Lyt-1+2-. Donor-type T cells in the spleen began to appear at about day 14 in chimeras that were transplanted with a larger dose of bone marrow cells, whereas this was slightly delayed in those grafted with a smaller dose of bone marrow cells, starting at about day 28.(ABSTRACT TRUNCATED AT 400 WORDS)

Strain difference in the radiosensitivity of immunocompetent cells and its influence on the residual host-vs-graft reaction in lethally irradiated mice grafted with semiallogeneic bone marrow.

A striking difference in radiosensitivity was noted between C3H/He (H-2k) and C57BL/6J (H-2b) strain mice when assessed by primary anti-SRBC PFC response of intact animals and primary cell-mediated lympholysis (CML) response of spleen cells to allogeneic cells in vitro, the C3H strain being more radioresistant. On the other hand, when C3H and B6 mice were exposed to 6.62 to 10.40 grays (Gy) of x-rays and then were transplanted with 2 X 10(6) bone marrow cells from B6C3F1 (H-2b/k) donor mice within 3 hr or at 24 hr after radiation exposure, the early mortality caused by residual host-vs-graft (HVG) reaction was much higher when C3H mice were used as recipients. Furthermore, the proportion of surviving animals manifesting host-type lymphohemopoiesis, i.e., host-type revertants, was much higher in B6C3F1 to C3H than in B6C3F1 to B6 combination. Spleen cells from such host-type revertants manifested strong anti-donor reactivity when assessed by mixed lymphocyte reaction (MLR) and/or CML in vitro. Increase of radiation doses to the recipients to 10.40 Gy resulted in 100% survival and 100% donor-type lymphohemopoiesis in both groups of chimeras. These results indicate strongly that a genetic difference in radiosensitivity of immune system of the recipients can greatly influence the magnitude of residual HVG reactions observed in hybrid to parental strain bone marrow transplantation in mice.

Anti-viral immune response of allogeneic irradiation bone marrow chimeras: cytotoxic T cell responsiveness depends upon H-2 combination and infectious agent.

Allogeneic irradiation bone marrow chimeras C57BL/10 (H-2b) leads to B10.BR (H-2k) and B10.BR (H-2k) leads to C57BL/10 (H-2b) were raised under specific pathogen-free (SPF) conditions; they survived very well and were healthy under SPF for 3-4 months and subsequently, under conventional housing conditions, for 1 to 8 months. Their immune response against third-party alloantigens was comparable with that of controls. Anti-vaccinia virus responses were very low when compared with syngeneic control chimeras or unmanipulated control animals; if anti-vaccinia virus cytotoxic T cell reactivity was measurable, it was specific for the bone marrow donor, rather than the recipient thymic H-2 type. In contrast, the anti-LCMV (lymphocytic-choriomeningitis virus) response was excellent and comparable to that in controls for B10.BR (H-2k) leads to C57BL/10 (H-2b) chimeras, but was completely absent for C57BL/10 (H-2b) leads to 10.BR (H-2k) chimeras. LCMV-specific cytotoxic effector T cells from B10.BR leads to C57BL/10 chimeras were restricted entirely to recipient H-2b. In contrast to the asymmetric cytotoxic T cell response, both types of chimeras developed good primary footpad swelling reactions after local infection, which arose somewhat slower with LCMV than in control of chimeras. The capacity to control infection by Listeria monocytogenes was excellent for all controls and B10.BR leads to C57BL/10 chimeras but apparently absent in C57BL/10 leads to B10.BR chimeras. Differentiation of T cell restriction specificity for thymic H-2 is apparently most efficient, but it remains unclear whether the observed asymmetry reflects exaggerated immune response regulation in H-2-incompatible stem cell-thymus chimeras or differential cross-reactivities between restricting transplantation antigens.