Nonirradiated Recipients Research Articles

Murine hematopoietic stem cell distribution and proliferation in ablated and nonablated bone marrow transplantation

The engraftment of donor bone marrow (BM) cells in nonablated mice is inefficient. Niche availability has been thought to be the reason, and cytoablation with irradiation or cytotoxic agents is routinely used with the belief that this frees the preoccupied niches in recipients. In this study, donor cell redistribution and proliferation in ablated and nonablated mice were compared by implanting donor cells directly into the femur cavity of sedated mice. The redistribution of Lin(-) donor cells into BM was similar between ablated and nonablated mice. Poor engraftment in nonablated mice was shown to be the result of inefficient donor cell proliferation rather than because of a lack of space. Competitive repopulation assays demonstrated that the donor hematopoietic stem cells (HSCs) were present in nonirradiated recipients for at least 6 months after transplantation, but that they did not expand as did their counterparts in lethally irradiated mice. This study suggests that efficient bone marrow transplantation in nonablated recipients may be possible as a result of better understanding of HSC proliferative regulation and appropriate in vitro manipulation.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo.

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%).

A Subset of CD8 Memory T Cells from Old Mice Have High Levels of CD28 and Produce IFN-γ

Using carboxyfluorescein diacetate succinimidyl ester (CFSE)-tagged cells to measure proliferation in vivo, we found that only memory CD8 + cells from mice older than 18 months gave measurable levels of proliferation and that the proportion of memory CD8 + T cells able to proliferate in a nonirradiated recipient increased with age. CD8 cells that had proliferated in vivo contained higher levels of CD28 when compared to CD8 cells that had not divided. Cells with high levels of CD28 were preferentially able to divide in nonirradiated recipients. Using ex vivo intracellular staining analysis, we determined that most of the CD8 + T cells that were capable of dividing in vivo produced IFN-γ after isolation from recipient mice or their original host. These studies thus document the presence in aged mice of a population of CD28 hi CD8 + cells whose ability to proliferate in vivo without antigenic stimulation and to produce IFN-γ may be involved in immune regulation.

Fibrin microbeads for isolating and growing bone marrow-derived progenitor cells capable of forming bone tissue.

It has been demonstrated that bone marrow (BM)-derived pluripotent stem cells can be incorporated into muscle, bone, nerve, lung, stomach, intestine, and skin. Fibrin-based biodegradable microbeads (FMB) were developed for culturing, in suspension, a high density of cells, mostly of mesenchymal origin. In the current study, FMB were used to isolate and expand mesenchymal progenitor cells from BM of mice and rats. Cells from BM isolated on FMB (FMB-BM cells) were visualized by fluorescent confocal microscopy and quantified by a modified MTS colorimetric assay. Downloading the BM cells from FMB onto plastic induced their differentiation into islets of cells with osteogenic phenotype that secreted mineralized extracellular matrix. This was augmented by inducers of osteogenesis, such as ascorbic acid, beta-glycerophosphate, and dexamethasone, or osteoblast-growth peptides (OGP). Implanting FMB-BM cells under the kidney capsule in mouse tested the osteogenic potential of these cells in vivo. Thirty days after implantation, bone structures with typical BM elements were seen in 8/53 kidneys in 6-Gy-irradiated mice and in 1/10 kidneys in nonirradiated recipients; bone formation was verified by soft x-ray imaging and elemental analysis that showed elevated Ca and Fe in the implant region. FMB-BM cells - downloaded onto plastic flasks, cultured for 2 weeks, mechanically harvested and then implanted - induced 100% bone formation in both irradiated (6/6) and nonirradiated (3/3) mice. Histology revealed well-organized bone structures under the kidney capsule, including osteoblasts and typical elements of BM. Our findings demonstrate that FMB are capable of isolating and expanding progenitor cells from BM for osteogenesis and possibly for regenerating other mesenchymal tissues.

Role of the passive apoptotic pathway in graft-versus-host disease.

Donor T cells have been shown to undergo apoptosis during graft-vs-host disease (GVHD). Although active apoptosis mediated through Fas/Fas ligand interactions has been implicated in GVHD, little is known about the role of the passive apoptotic pathway. To examine this question, we compared the ability of normal donor T cells and T cells overexpressing the antiapoptotic protein, Bcl-x(L), to mediate alloreactive responses in vitro and lethal GVHD in vivo. In standard MLCs, T cells that overexpressed Bcl-x(L) had significantly higher proliferative responses but no difference in cytokine phenotype. Overexpression of Bcl-x(L) prolonged survival of both resting and alloactivated CD4(+) and CD8(+) T cells as assessed by quantitative flow cytometry, accounting for the higher proliferative responses. Analysis of engraftment in murine transplantation experiments demonstrated an increase in donor T cell chimerism in animals transplanted with Bcl-x(L) T cells, suggesting that overexpression of Bcl-x(L) prolonged T cell survival in vivo as well. Notably, transplantation of Bcl-x(L) T cells into nonirradiated F(1) recipients also significantly exacerbated GVHD as assessed by mortality and pathological damage in the gastrointestinal tract. However, when mice were irradiated no difference in GVHD mortality was observed between animals transplanted with wild-type and Bcl-x(L) T cells. These data demonstrate that the passive apoptotic pathway plays a role in the homeostatic survival of transplanted donor T cells. Moreover, the susceptibility of donor T cells to undergo passive apoptosis is a significant factor in determining GVHD severity under noninflammatory but not inflammatory conditions.

Personal pathways to embryonic stem cells

Work with numerous colleagues on embryo stem cells in vitro began in our laboratories in 1963, using cleaving embryos and blastocysts of the rabbit. Growth of disaggregated blastomeres from cleavage-stage embryos was weak. Trophoblast outgrowths from whole-embryo cultures provided a platform supporting inner cells that differentiated into blood islands, muscle, nerve cells, phagocytic cells, connective tissue and undefined elements. Stem cell lines derived from whole or disaggregated cells of rabbit inner cell mass and embryonic disc proved to be long-lasting (immortal), growing over months and years, stable enzymically, karyotypically and morphologically, and fully capable of resuming growth after cryopreservation. To measure the embryological potency of embryo stem cells, disaggregated or entire inner cell masses were injected into the blastocoelic cavity of mouse blastocysts. They contributed to the formation of chimaeras, and partially colonized most or all recipient tissues except trophectoderm. Gene markers for coat colour enabled their instant detection in recipient fetuses, newborns and adults. Cell lineages, the multipotency of single cells, and transgenesis all descended from this approach. Mouse and rat stem cells were grown from disaggregated blastocysts cultured in vitro or from tissues extracted from early post-implantation mouse embryos. They fully recolonized bone marrow in lethally irradiated adult mouse recipients, and in mice carrying inherited anaemias, migrating via liver to bone marrow and spleen. Some may have been hepatocyte or splenocyte precursors. Non-irradiated recipients were weakly colonized by embryo stem cells overcoming histocompatibility barriers. No signs of inflammation or cancer were noted. First studies on human embryo stem cells began, but were ended by an ethical decision preventing supplies of human blastocysts for research.

Homing of primitive hematopoietic cells in non-myeloablated murine recipients

Investigations into the homing of hematopoietic stem cells (HSC) following transplantation have most commonly been performed in myeloablated recipients. Recent success of bone marrow (BM) transplantation in non- or minimally-myeloablated recipients has increased interest in examining homing patterns of HSC in non/myeloablated hosts. To this end, low-density murine BM cells were stained with PKH2 and transplanted into lethally-irradiated (18 hr post-irradiation) or non-irradiated syngeneic recipients, which were sacrificed at various time points post-transplantation (PT). Cells harvested from the peripheral blood (PB), BM, and spleen were analyzed for recovery of donor cells, adhesion molecule profile, cell cycle status, and proliferation history. Despite relative enrichment of donor cells in myeloablated tissues, recovery from all 3 tissues of donor cells up to 24 hr PT was similar between myeloablated and non-ablated recipients. Recovery ranged from 6 to 10% of the original graft, with 60–90% of the recovered cells homing to BM. Recovery of donor Sca-1+ lin− cells from BM was up to 8-fold higher than recovery total donor cells in both transplant settings, suggesting specific homing of primitive phenotypes. Interestingly, recovery of Sca-1+ lin− cells was greater in non-myeloablated recipients compared to irradiated recipients. Analysis of cell cycle status of donor cells recovered from BM, spleen, or PB revealed a high degree of quiescence, while BRD-U incorporation indicated that 10 to 30% of these cells had cycled in vivo by 24 hr PT. Examination of adhesion molecule expression of BM-homed Sca-1+ cells revealed that phenotypes enriched for long-term engraftment potential, such as Sca-1+ lin− CD43+ cells, homed preferentially to the marrow of non-irradiated recipients compared to that of irradiated hosts. These data suggest that while homing in myeloablated recipients may represent a random process due to radiation damage of stroma and/or endothelial cells, homing in non-myeloablated recipients may better portray natural trafficking patterns of HSC in vivo.

Lymphoma cell contamination of PBSC: a murine model.

In P-388 bearing BDF1 mice stem cell mobilisation was tested on the survival of lethally irradiated isologous recipients. Contamination of the graft with lymphoma cells was evaluated by the number of clonogenic lymphoma cells (CFU-L) and the ability of the graft to induce lymphoma in non-irradiated recipients. A mobilisation protocol (200 mg/kg cyclophosphamide (CY) i.p. or 200 mg/kg CY followed by 125 microg/kg G-CSF administered every 12 h for 3 consecutive days, starting 14 days after lymphoma initiation) that resulted in a substantial stem cell mobilisation in normal mice, mobilised too few CFU-L to induce lymphoma in the recipients: 50 microl of blood obtained after mobilisation protected lethally irradiated mice but did not induce lymphoma in normal recipients. A minimum graft of bone marrow (2 x 105 cells, with 5580 CFU-L) from untreated P-388 bearing donors killed irradiated recipients presumably by lymphoma and not bone marrow failure. The mobilisation protocol reduced CFU-L so much that no lymphoma-associated death occurred even after the transplantation of 10 x 105 bone marrow cells. These data suggest that, although the PBSC mobilisation protocol may also mobilise some clonogenic lymphoma cells, with the minimum graft size no lymphoma transfer can be observed.

Behavioural consequences of oligodendrocyte progenitor cell transplantation into experimental demyelinating lesions in the rat spinal cord.

Glial cell transplantation has the potential to be developed into a clinical treatment for human demyelinating diseases because of its demonstrated efficacy in remyelinating experimentally demyelinated axons. As a step towards clinical application it is necessary to demonstrate that the procedure is safe and efficacious in promoting behavioural recovery. In this study we transplanted glial cell progenitors into demyelinating lesions induced by intraspinal injection of ethidium bromide in the rat. Locomotor function after transplantation was assessed using a beam-walking test that has previously been shown able to detect deficits associated with demyelination in the dorsal funiculus of the rat spinal cord. Two groups of animals with transplants were examined. In one group, spontaneous remyelination was prevented by exposure of the lesion to 40 Gy of X-irradiation; in the other, male glial cells were transplanted into nonirradiated female recipients, permitting their identification by use of a probe specific to the rat Y chromosome. Following transplantation, there was severe axon loss in a large proportion of the irradiated animals and those affected did not recover normal behavioural function. In contrast, both the small proportion of the irradiated group that sustained only mild axon loss and the nonirradiated recipients of transplants recovered normal function on our behavioural test. We conclude that glial cell transplantation is able to reverse the functional deficits associated with demyelination, provided axonal loss is minimal.

Hemopoietic microenvironment-transferring units and inducible hemopoietic stromal precursors in the bone marrow of thymectomized mice

The method of heterotopic transplantation of the bone marrow was used to study the effect of thymectomy on clonogenic and inducible hemopoietic stromal precursors in adult rats. The self-maintenance or clonogenic capacity of stromal precursors was evaluated by retransplantation of primary hemopoietic foci. The kinetics of the formation of ectopic foci from thymectomized rats is similar to that of normal bone marrow. The presence of inducible stromal hemopoietic precursors was evaluated by the stimulation index (the ratio of the size of hemopoietic focus formed in irradiated to that in nonirradiated recipient). It is found that the growth of ectopic focus in chimeras is stimulated by a nonthymic factor, which suggests thymus-independent regulation of hemopoietic microenvironment precursor cells.

Preleukemia in long-term plasmacytoma-regressor mice

Our previous results have indicated that mice whose plasmacytoma regressed following curative melphalan chemotherapy manifested various persistent immunohematological abnormalities including immunosuppression, myeloproliferation, as well as excessive production of and response to growth factors. Mice not bearing plasmacytoma treated with an identical dose of melphalan chemotherapy did not exhibit such abnormalities. In the present study we show that plasmacytoma-regressor mice (PRM) contain preleukemic cells which do not progress to leukemia in these mice. However, adoptive transfer of splenocytes originating in PRM to preirradiated but otherwise untreated syngeneic recipients resulted in the development of overt leukemia in these recipients. The presence of leukemia in the primary recipient mice was ascertained by blood counts as well as by spleen histology. Furthermore, splenocytes from the irradiated primary recipients adoptively transferred to non-irradiated secondary recipients caused leukemia formation in 100% of the secondary recipients. Sex chromosome analysis of the leukemic cells in the irradiated primary recipients clearly showed that they originated in the PRM donors. Two leukemic lines were established from leukemias developing in the secondary recipients and both expressed surface markers of hematopoietic progenitor cells as well as markers of T cells. We suggest that PRM could serve as an animal model to investigate development of chemotherapy-related leukemia in humans.

Preleukemia in long-term plasmacytoma-regressor mice.

Our previous results have indicated that mice whose plasmacytoma regressed following curative melphalan chemotherapy manifested various persistent immunohematological abnormalities including immunosuppression, myeloproliferation, as well as excessive production of and response to growth factors. Mice not bearing plasmacytoma treated with an identical dose of melphalan chemotherapy did not exhibit such abnormalities. In the present study we show that plasmacytoma-regressor mice (PRM) contain preleukemic cells which do not progress to leukemia in these mice. However, adoptive transfer of splenocytes originating in PRM to preirradiated but otherwise untreated syngeneic recipients resulted in the development of overt leukemia in these recipients. The presence of leukemia in the primary recipient mice was ascertained by blood counts as well as by spleen histology. Furthermore, splenocytes from the irradiated primary recipients adoptively transferred to non-irradiated secondary recipients caused leukemia formation in 100% of the secondary recipients. Sex chromosome analysis of the leukemic cells in the irradiated primary recipients clearly showed that they originated in the PRM donors. Two leukemic lines were established from leukemias developing in the secondary recipients and both expressed surface markers of hematopoietic progenitor cells as well as markers of T cells. We suggest that PRM could serve as an animal model to investigate development of chemotherapy-related leukemia in humans.

Small cortical thymocytes are subject to positive selection.

To determine the developmental stages at which positive selection can act to produce mature T cells, CD4+8+3lo thymocytes of large dividing type and of small nondividing type were sorted and transferred into the thymus of nonirradiated Thy-1 congenic recipient mice. In contrast to earlier studies, the small as well as the large thymocytes produced mature CD4+8-3hi and CD4-8+3hi progeny, although production was less efficient from the small cells. The relative efficiency of small cells was increased and was close to that of large cells when bcl-2/anti-HY T cell receptor (TCR) alpha beta transgenic donors were used to improve cell survival, overcome stress effects of the transfer process, and increase the frequency of selectable cells. The results from transferring small CD4+8+3lo thymocytes expressing a TCR transgene from a nonselecting to a selecting thymic MHC environment also confirmed that the small cells were capable of being selected and maturing. Thus the developmental window available for positive selection includes the small CD4+8+3lo thymocytes. The results also showed a striking difference in the kinetics of production of mature progeny from the transferred CD4+8+3lo precursors. CD4+8-3hi cells appeared several days before CD4-8+3hi cells, apparently because the CD4-8+ lineage cells spent several days in transit as CD4+8+3hi intermediates before losing CD4. Most CD4+8- lineage cells on the other hand, either passed very rapidly through this intermediate stage, or lost CD8 before increasing the expression of CD3.

Evidence of CD4+ regulatory T cells in the non-obese diabetic male mouse

The NOD mouse, which shows many features of human IDDM, is extensively used to evaluate the role of T lymphocytes in the pathogenesis of autoimmune diabetes. The development of diabetes in this model appears to be controlled by a finely tuned immunoregulatory balance between autoaggressive T cells and regulatory immune phenomena, the disruption of which may result in destruction of insulin-secreting cells. The absolute requirement of sublethal irradiation to permit transfer of the disease to non-diabetic adult syngeneic mice provides indirect evidence for the presence of regulatory T cells in non-diabetic NOD mice. We have previously reported that the reconstitution of irradiated recipients by CD4 + T cells from nondiabetic female NOD mice blocks the transfer of diabetes by spleen cells from diabetic donors. We now report evidence that anti-CD4 monoclonal antibodies can substitute for irradiation in rendering adult NOD male mice susceptible to diabetes transfer by diabetogenic spleen cells. Efficient diabetes transfer can be achieved in non-irradiated adult NOD recipients provided they are thymectomized and CD4 + T-cell depleted prior to the transfer. The role of thymectomy is to limit T cell regeneration after anti-T cell monoclonal antibody challenge. Our data confirm that regulatory CD4 + T-cells, which efficiently counterbalance diabetogenic cells, are present in adult NOD male animals.

Evidence of CD4+ regulatory T cells in the non-obese diabetic male mouse.

The NOD mouse, which shows many features of human IDDM, is extensively used to evaluate the role of T lymphocytes in the pathogenesis of autoimmune diabetes. The development of diabetes in this model appears to be controlled by a finely tuned immunoregulatory balance between autoaggressive T cells and regulatory immune phenomena, the disruption of which may result in destruction of insulin-secreting cells. The absolute requirement of sublethal irradiation to permit transfer of the disease to non-diabetic adult syngeneic mice provides indirect evidence for the presence of regulatory T cells in non-diabetic NOD mice. We have previously reported that the reconstitution of irradiated recipients by CD4+ T cells from nondiabetic female NOD mice blocks the transfer of diabetes by spleen cells from diabetic donors. We now report evidence that anti-CD4 monoclonal antibodies can substitute for irradiation in rendering adult NOD male mice susceptible to diabetes transfer by diabetogenic spleen cells. Efficient diabetes transfer can be achieved in non-irradiated adult NOD recipients provided they are thymectomized and CD4+ T-cell depleted prior to the transfer. The role of thymectomy is to limit T cell regeneration after anti-T cell monoclonal antibody challenge. Our data confirm that regulatory CD4+ T-cells, which efficiently counterbalance diabetogenic cells, are present in adult NOD male animals.

Immunosuppression by lymphocytic choriomeningitis virus infection: competent effector T and B cells but impaired antigen presentation.

Lymphocytic choriomeningitis virus (LCMV) may cause a severe immunosuppression in mice. Its pathogenesis is apparently dependent on LCMV-specific CD8 effector T cells that mediate the destruction of virus-infected cells which are normally essentially involved in immune responses. Evaluation of various LCMV isolates in this study established a general correlation between their tropism for lymphohemopoietic cells and immunosuppression. When immune responses were assessed as the capacity of mice to mount an anti-vaccinia virus cytotoxic T cell response or an IgG response to vesicular stomatitis virus (VSV), after a primary LCMV infection, LCMV-Armstrong, WE, Clone 13 and Docile were increasingly immunosuppressive in a dose-dependent fashion with respect to both extent and duration. Analysis of lymphocyte subpopulations showed variable effects of the various LCMV isolates that did not reveal patterns readily explaining immunosuppression. To evaluate whether LCMV infection affected T and/or B cell functions directly or whether antigen presentation was impaired, adoptive transfer experiments were performed. Untreated or irradiated but uninfected normal recipient mice receiving adoptively transferred T or B cells from LCMV-WE or Docile-infected immunosuppressed donor mice responded within 30%-100% of normal ranges in both assay systems. In contrast, when T or B cells from normal donors were transferred to irradiated or non-irradiated LCMV-immunosuppressed recipients, they failed to mount a significant cytotoxic T cell response against vaccinia virus or an IgG response to VSV. Thus, the T and B cells from LCMV-immunosuppressed mice were able to function within normal ranges; in contrast, histologically and functionally, antigen presentation was severely impaired in LCMV-immunosuppressed mice.

Selective localisation of neuro-specific T lymphocytes in the central nervous system

Using experimental autoimmune encephalomyelitis (EAE) in the rat as a model of central nervous system (CNS) inflammation, activated and quiescent T lymphocytes with different antigen specificities were labelled with the fluorescent dye Hoechst 33342 and tested by fluorescence microscopy for their ability to accumulate in different regions of the spinal cord and in other organs at varying times post inoculation. With this highly sensitive assay it was found that activated myelin basic protein (MBP)-specific T cell lines accumulated in the spinal cord (a 1000-fold increase in the lumbar/sacral region by day 4) and caused clinical signs of EAE. In contrast, interleukin-2 (IL-2)-maintained (quiescent) MBP-specific T cell lines failed to accumulate in the CNS and cause disease. Activated ovalbumin (OA)-specific and purified protein derivative of tuberculin (PPD)-specific T cell lines were also found at significantly higher levels in the spinal cord than non-activated cells although they failed to accumulate to a substantial degree when injected alone. When injected with activated MBP-specific T cells the activated OA- and PPD-specific cell lines accumulated in the spinal cord following initial accumulation of the MBP-specific cells, demonstrating that during the inflammatory process there is considerable non-specific recruitment of cells into the inflammatory site. CNS accumulation of activated MBP-specific T cell liness occurred 1–2 days later in irradiated animals than in non-irradiated recipients. This was consistent with irradiated animals also exhibiting a later onset of disease and suggests that irradiation may directly affect the endothelium in a way that makes it less adhesive. In conclusion, this study demonstrates that activated lymphocytes of any specificity enter the spinal cord, and that the neuro-antigen specific cells accumulate there and lead to the recruitment of other cells. Non-activated cells, even those with neural antigen specificity fail to enter the cord. Understanding the nature of what an ‘activated’ lymphocyte is may allow us to design strategies to inhibit such immune-mediated inflammation.

Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease.

In this report we have investigated macrophage (M phi) activity and tumor necrosis factor alpha (TNF-alpha) production during graft-vs.-host disease (GVHD). TNF-alpha production by M phi requires two signals: priming of M phi by interferon followed by triggering of TNF-alpha production and release by lipopolysaccharide (LPS). The state of M phi activation was examined in nonirradiated B6AF1 recipient mice injected with either 60 x 10(6) (acute GVHD) or 30 x 10(6) (nonlethal GVHD) parental B6 lymphoid cells. During the early phase of acute GVHD, administration of normally sublethal amounts of LPS-triggered release of significant amounts of TNF-alpha into the serum resulting in death of the animals within 36 h. Normal animals treated with the same dose of LPS neither died nor produced detectable amounts of serum TNF-alpha. In vitro studies demonstrated that M phi were primed during GVHD. The level of M phi priming was greater during acute GVHD than nonlethal GVHD since 100-fold less LPS was required to trigger killing of a TNF-alpha-sensitive cell line by M phi from acute GVHD animals. The amount of TNF-alpha released into the serum after LPS injection increased during the course of the GVHD and was significantly greater in acute GVH-reactive mice. Endogenous LPS was detected in the serum of acute GVH-reactive animals coincident with the onset of mortality. The data provide evidence that during GVHD M phi are primed as a result of the allogeneic reaction and that endogenous LPS therefore triggers M phi production of TNF-alpha resulting in the symptoms characteristic of acute GVHD.

Chronic myeloproliferative disease induced by site-specific integration of Abelson murine leukemia virus-infected hemopoietic stem cells.

We recently showed that hemopoietic stem cells expressing the v-abl oncogene can cause leukemia when injected into lethally irradiated recipient mice. Progenitor cells expressing v-abl did not significantly contribute to disease development, and the leukemia was monoclonal in origin. By serially transplanting v-abl-transduced hemopoietic stem cells into normal, nonirradiated syngeneic recipients, we showed that multiple stem-cell clones do exist in some recipients. These cells fluctuated as normal stem cells do and could home to normal bone marrow. Based on the time course of disease, the recipients developed either an acute or a chronic phase of disorder. All recipients with the acute disease had stem-cell clones with random Abelson murine leukemia virus integration sites. All recipients with the chronic disorder had a specific Abelson murine leukemia virus integration site. We believe this abl-specific integration site, termed ASI, is important in abl-mediated stem-cell leukemogenesis.

Autoimmune response induction and regulation in rat erythrocyte-immunized mice

The process of antibody formation to self-red blood cells (RBC) has been studied in rat RBC (rRBC)-immunized mice. A positive correlation was noted between antibody production to mouse RBC (mRBC) and rRBC in some mouse strains. The low responsiveness on both indices was overcome by s.c. injections of rRBC in low doses. rRBC-tolerant mice exhibited lower levels of antibody production to mRBC. Splenocytes from rRBC-immunized donors, when transferred to irradiated recipients, revealed enhanced and accelerated anti-mRBC and anti-rRBC antibody production in response to rRBC but not to autologous mRBC. Consequently, the autoimmune process is not accompanied by disordered immunologic tolerance to self-RBC and requires participation of Th responding to foreign epitopes of rRBC antigens. Splenocytes from rRBC-immunized donors, when transferred to non-irradiated recipients, inhibited antibody production to mRBC. The suppressive effect was not abrogated by pretreating donors or recipients with low doses of cyclophosphamide (CP) or by pretreating donors with antibodies to I-J. It was abrogated by the elimination of cells of donor origin within 8-9 days after transfer. Inoculation of antibodies to rRBC in immunized mice on a schedule imitating their splenocyte transfer dynamics inhibited antibody production to mRBC. Therefore, it can be assumed that the suppressive effect of cell transfer is accounted for, not by suppressors or their inducers, but by antibodies to rRBC on the basis of feedback regulation.