THE ROLE OF BONE MARROW OF X-IRRADIATED MICE IN THYMIC RECOVERY

The influence of the bone marrow on the repopulation of the thymus in X-irradiated mice has been investigated.
It was observed that the thymus and a certain population of bone marrow lymphocytic cells were repopulated in parallel in a cyclic fashion. This occurred either after a single exposure of mice to 400 R or after serial weekly X-ray treatments with 170 R. Lethally irradiated recipients which were grafted with bone marrow cells obtained 12-24 days after four weekly irradiations of donor mice with 170 R also exhibited a cyclic repopulation of both the thymus and the bone marrow lymphocytic population. In contrast, mice which were transplanted with bone marrow cells from unirradiated donors, containing an equal number of stem cells (CFU), exhibited a continuous rather than a cyclic recovery of both cell populations. the bone marrow stem cells of mice recovering from X-irradiation were found to have a decreased proliferative activity, since they produced significantly smaller spleen colonies in lethally irradiated recipients than marrow cells from unirradiated mice.
The results were interpreted as indicating that the bone marrow lymphocytic cells may act as thymic precursor cells and that thymic lymphopoiesis is dependent on the presence of such cells. Evidently, the production of lymphocytic cells will decrease when the stimulus for granulocyte production increases due to the limited proliferative activity of the surviving bone marrow stem cells after irradiation. This may result in a cyclic variation of the production of bone marrow lymphocytic cells and it follows that thymic lymphopoiesis will run parallel.     

Influence of thymectomy on the lymphoid regeneration of hematopoietic bone marrow after sublethal irradiation]

In C57BL mice, bone marrow lymphoid regeneration after a sublethal irradiation is modified by a graft of normal marrow cells. This effect is suppressed in thymectomized mice since a lymphoid peak is observed after a 350 R irradiation; its composition is heterogeneous: small lymphocytes, lymphoblasts and peculier cells named "X cells". The same phenomenon is observed in mice where all the thymocytes and thymus derived and peripheral lymphocytes are destroyed. These results exclude that bone marrow lymphoid regeneration after irradiation is due to a migration of lymphoid cells of thymic origin to the marrow. They could be explained by the effect of a humoral thymic factor on marrow lymphopoiesis.     

Suppression by bone marrow cells of the level of specific IgE antibodies in the blood of mice and decrease in the production of IGE by bone marrow cells

Changes in the level of the specific IgE-antibodies to ovalbumin under the influence of syngeneic cells of a bone marrow were studied. The IgE-response was induced by ovalbumin in mice (CBA X C57Bl/6)F1. The bone marrow cells suspensions (20-30 X 10(6) cells per mouse) from syngeneic donors was inoculated simultaneously with the immunization. It was found that bone marrow cells suppressed both the level of IgE-antibodies in experimental mice serum and the production of IgE by the bone marrow cells of the recipient. The ability to suppress IgE-response remained when erythrocytes, monocytes and T-lymphocytes were removed from inoculated suspensions. The bone marrow cells taken from the mice immunized with ovalbumin, at the stage of a decreasing IgE-response, provided more pronounced suppression, than bone marrow cells taken from intact animals.     

Mouse skin graft prolongation with donor strain bone marrow and anti-lymphocyte serum: surface markers of the active bone marrow cells

The survival of C3H/HeJ skin grafts on B6AF1 mice treated with anti-lymphocyte serum (ALS) can be significantly prolonged by the injection of the host with C3H/HeJ bone marrow. Although the prolongation is apparently due at least in part to the ultimate presence in the host of specific suppressor cells of donor origin, little is known about the nature of the cells in the marrow inoculum that are responsible for this effect. The present investigation was undertaken to characterize surface markers of the active bone marrow cells. Marker-positive populations were either depleted and enriched by panning techniques or depleted by killing with specific antibody and complement, and then were assayed for their ability to prolong graft survival. Cells that were adherent to anti-Ia-coated plates extended graft survival only slightly better than did treatment with ALS alone, whereas nonadherent (Ia-depleted) cells, as well as cells treated with anti Ia and complement, retained good prolonging activity. Similarly, panning on anti-immunoglobulin (Ig)-coated plates produced an active, Ig+-depleted population and an inactive adherent population, and killing of Thy-1+ cells with antibody and complement did not compromise the ability of the bone marrow inoculum to prolong graft survival. Complement receptor-positive (EAC+) and Fc gamma receptor-positive cells (EA+) were separated by panning on plates coated with sheep erythrocytes/antibody/complement and erythrocytes/7S antibody respectively. Adherent, EAC+-enriched cells were only slightly active, whereas the nonadherent, EAC-depleted population was fully active in graft prolongation. However, both Fc gamma R+ (EA+)-enriched and depleted populations were active, with the enriched fraction producing significantly better prolongation than the depleted population. Thus, the bone marrow cells that can prolong skin graft survival in ALS-treated mice appear to be Ia-, Thy-1+, largely complement receptor negative, and Ig-, but are largely positive for Fc gamma receptors.     

Studies on the recovery from tolerance to tumor antigens

Summary The present study investigates the potential of bone marrow cells from mice tolerant to tumor antigens to repopulate tumor-specific effector T cells. C3H/He mice were inoculated i.v. with 10⁶ 10000 R X-irradiated syngeneic X5563 plasmacytoma tumor cells three times at 4-day intervals. This regimen abrogated the ability of spleen cells from these mice to develop anti-X5563 cytotoxic and in vivo protective (tumor-neutralizing) T cell-mediated immunity as induced by i.d. inoculation of viable X5563 cells followed by surgical resection of the tumor. Since such suppression was induced in a tumor-specific way, this represented a state of antitumor tolerance. When bone marrow cells from normal or X5563-tolerant mice were transferred i.v. into 950 R X-irradiated syngeneic C3H/He mice, both groups of recipient mice generated anti-X5563 tumor immunity over a similar time course and to almost the same degree. Anti-X5563 tumor immunity induced in (C3H/He×C57BL/6) F1 mice which had been transferred with bone marrow cells from normal or X5563-tolerant C3H/He mice were mediated by T cells expressing the Ly phenotype of C3H/He, but not of C57BL/6, excluding the possibility that the antitumor effector cells were derived from recipient mice. It was also demonstrated that C3H/He mice which had been reconstituted with normal marrow were rendered tolerant when the tolerance regimen was started 7 weeks, but not 1 week after the bone marrow reconstitution. These results indicate that bone marrow cells from antitumor tolerant mice are not rendered tolerant to the tumor but can provide the potential to repopulate antitumor CTL and in vivo protective effector T cells.This work was supported by the Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan Abbreviations used: MHC, major histocompatibility complex; CTL, cytotoxic T lymphocytes; TNP, trinitrophenyl; C, complement; TNBS; trinitrobenzene sulfonate; MMC, mitomycin C     

Migration of natural suppressor cells from bone marrow to peritoneal cavity by live BCG

Delayed-type hypersensitivity (DTH) responses were suppressed in mice inoculated with bone marrow cells from mice that had been injected with 10(8) colony-forming units (CFU) of live BCG. Upon analysis of this DTH-suppression by the use of a macrophage migration inhibition (MI) assay, the in vitro correlate of DTH, suppressor macrophages in the peritoneal cavity were found to play an important role in DTH suppression. However, neither suppression of DTH nor production of suppressor macrophages was observed in mice inoculated with bone marrow cells from mice that had been injected with methotrexate (MTX), a folic acid antagonist, and 10(8) CFU of live BCG. Moreover, suppressor cells against the MI activity of peritoneal exudate cells from BCG cell wall-immunized mice existed in bone marrow cells from normal mice, natural suppressor (NS) cells, and they were sensitive to MTX. In addition, these NS cells phagocytized carbonyl iron particles, were adherent to Sephadex G-10, and had Fc receptors, but they had no B or T cell markers, suggesting that these cells belonged to a macrophage compartment. From this evidence, we hypothesized that the origin of suppressor macrophages in the peritoneal cavity induced by live BCG injection was MTX-sensitive NS cells in bone marrow, and that these NS cells were stimulated by a small dose of live BCG trapped in bone marrow after i.v. injection of a high dose of live BCG and migrated from bone marrow to the peritoneal cavity.     

Induction of bone marrow allograft rejection and hybrid resistance in nonresponder recipients by antibody: is there evidence for a dual receptor interaction in acute marrow graft rejection?

Acute marrow graft rejection in allogeneic or semiallogeneic donor-recipient mouse combinations has been suggested to be caused by natural killer (NK) cells. The unique in vitro specificity of NK cells for tumor cells, however, does not explain the specific rejection of bone marrow grafts by NK cells. Recent experiments have implicated antibody in marrow graft recipients as the specificity-inducing component that guides NK cells in an antibody-dependent cytotoxic (ADCC) reaction to attack the marrow graft. On the basis of this hypothesis, one would postulate that nonresponder marrow graft recipients can be converted into responders by injection with antibody of appropriate specificity. Results presented in this report show that this is indeed possible. Specific monoclonal or polyclonal antibody of IgG isotype induces marrow graft rejection in nonresponder recipients. This can be demonstrated in allogeneic as well as in semi-allogeneic (hybrid resistance) donor-recipient strain combinations. Antibody-induced marrow graft rejection is independent of complement and dependent on the presence of NK cells. Surprisingly, graft rejection induced by antibody is quite efficient in allogeneic and semiallogeneic marrow donor-recipient combinations, whereas it is generally poor in syngeneic combinations. This result is not understood if NK cells lyse bone marrow cells solely in an ADCC-type reaction. Because NK cells can lyse targets in an antibody-dependent as well as independent reaction, it is proposed that the binding of NK cells to targets via their receptors plays an additional role in the rejection of bone marrow in vivo. Preliminary evidence for this possibility is that NK cells in the apparent absence of antibody may have a detectable suppressive effect on the growth of marrow grafts in F1 hybrid mice transplanted with parental marrow grafts.     

Abrogation of hybrid resistance to bone marrow engraftment by graft-vs-host-induced immune deficiency

Lethally irradiated F1 mice, heterozygous at the hematopoietic histocompatibility locus Hh-1, which is linked with H-2Db, reject bone marrow grafts from H-2b parents. This hybrid resistance (HR) is reduced by prior injection of H-2b parental spleen cells. Because injection of parental spleen cells produces a profound suppression of F1 immune functions, we investigated whether parental-induced abrogation of HR was due to graft-vs-host-induced immune deficiency (GVHID). HR was assessed by quantifying engraftment of H-2b bone marrow in F1 mice with the use of splenic [125I]IUdR uptake; GVHID, by the ability of F1 spleen cells to generate cytotoxic T lymphocytes (CTL) in vitro. We observed a correlation in the time course and spleen cell dose dependence between loss of HR and GVHID. Both GVHID and loss of HR were dependent on injection of parental T cells; nude or T-depleted spleen cells were ineffective. The injection of B10 recombinant congenic spleens into (B10 X B10.A)F1 mice, before grafting with B10 marrow, demonstrated that only those disparities in major histocompatibility antigens that generated GVH would result in loss of HR. Thus, spleens from (B10 X B10.A(2R]F1 mice (Class I disparity only) did not induce GVHID or affect HR, whereas (B10 X B10.A(5R))F1 spleens (Class I and II disparity) abrogated CTL generation and HR completely. GVHID produced by a class II only disparity, as in (B10 X B10.A(5R))F1 spleens injected into (B6bm12 X B10.A(5R))F1 mice, was also sufficient to markedly reduce HR to B10 bone marrow. This evidence that GVHID can modulate hematopoietic graft rejection may be relevant to the mechanisms of natural resistance to marrow grafts in man.     

Epstein-Barr virus induces erosive arthritis in humanized mice

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of rheumatoid arthritis (RA) on the basis of indirect evidence, such as its presence in affected joint tissues, antigenic cross reactions between EBV and human proteins, and elevated humoral and cellular anti-EBV immune responses in patients. Here we report development of erosive arthritis closely resembling RA in humanized mice inoculated with EBV. Human immune system components were reconstituted in mice of the NOD/Shi-scid/IL-2Rγ(null) (NOG) strain by transplantation with CD34(+) hematopoietic stem cells isolated from cord blood. These humanized mice were then inoculated with EBV and examined pathologically for the signs of arthritis. Erosive arthritis accompanied by synovial membrane proliferation, pannus formation, and bone marrow edema developed in fifteen of twenty-three NOG mice transplanted with human HSC and inoculated with EBV, but not in the nine NOG mice that were transplanted with HSC but not inoculated with EBV. This is the first report of an animal model of EBV-induced arthritis and strongly suggest a causative role of the virus in RA.     

Characterization of effector cells of graft vs leukemia following allogeneic bone marrow transplantation in mice inoculated with murine B-cell leukemia

Summary It is now widely accepted that immunocompetent lymphocytes in allogeneic bone marrow grafts exert an antileukemic effect that contributes to the cure of leukemia. Graft vs leukemia (GVL) effects independent of graft vs host disease were investigated in allogeneic bone marrow chimeras tolerant of host and donor alloantigens. The role of Thy1.2, L3T4 and Lyt2 T lymphocytes as effector cells of GVL were investigated in (BALB/c × C57BL/6)F1 mice inoculated with murine B-cell leukemia and subsequently conditioned with total lymphoid irradiation and cyclophosphamide (200 mg/kg). Mice were reconstituted with C57BL/6 bone marrow cells depleted of well-defined T-cell subsets or enriched for stem cells by the soybean agglutination method. Detection of residual tumor cells, an indicator for efficacy of GVL, was carried out by adoptive transfer of peripheral blood or spleen cells obtained from treated chimeras into secondary naive BALB/c recipients at different time intervals following bone marrow transplantation. Treatment of the primary marrow inoculum with monoclonal anti-Thy 1.2 or anti-Lyt2 abolished the GVL effects and all secondary BALB/c recipients developed leukemia within 60 days. On the other hand, the treatment with monoclonal anti-L3T4 did not influence the effect of GVL and all treated recipients remained without leukemia. The data suggest that T cells may mediate GVL effects in the absence of graft vs host disease and in circumstances where tolerance to conventional alloantigens is elicited. Effector cells of GVL across the major histocompatibility complex (MHC) in the murine B-cell leukemia tumor model system appear to be Thy 1.2⁺ Lyt2⁺ L3T4—. Induction of GVL effects by allogeneic cells tolerant of host MHC suggests that these effects may be independent of graft vs host disease.     

Bone marrow-derived T lymphocytes responsible for allograft rejection

Lethally irradiated mice reconstituted with syngeneic bone marrow cells were grafted with allogeneic skin grafts 6-7 weeks after irradiation and reconstitution. Mice with intact thymuses rejected the grafts whereas the mice thymectomized before irradiation and reconstitution did not. Thymectomized irradiated mice (TIR mice) reconstituted with bone marrow cells from donors immune to the allografts rejected the grafts. Bone marrow cells from immunized donors, pretreated with Thy 1.2 antibody and C', did not confer immunity to TIR recipients. To determine the number of T lymphocytes necessary for the transfer of immunity by bone marrow cells from immunized donors, thymectomized irradiated mice were reconstituted with nonimmune bone marrow cells treated with Thy 1.2 antibody and C' and with various numbers of splenic T lymphocytes from nonimmune and immune donors. Allogeneic skin graft rejection was obtained with 10(6) nonimmune or 10(4) immune T cells. The effect of immune T cells was specific: i.e., immune T cells accelerated only rejection of the relevant skin grafts whereas against a third-party skin grafts acted as normal T lymphocytes.     

Allogeneic chimerism in scid mice after neonatal transfer of bone marrow.

Allogeneic chimeras are valuable tools for studies of complex immune cell interactions in vivo. Mice with severe combined immune deficiency (scid) should be ideal hosts for chimerism with allogeneic bone marrow cells as these animals lack mature T and B lymphocytes capable of reacting against donor alloantigens. However, it has been difficult to achieve full reconstitution of adult scid mice even using coisogenic bone marrow grafts without prior irradiation of the recipient. We explored ways to generate complete reconstitution of scid mice with allogeneic bone marrow. Unirradiated adult scid recipients of allogeneic bone marrow were only marginally reconstituted. Adult scid mice pretreated with 250 R were reconstituted with allogeneic bone marrow as measured by serum IgM concentration, peripheral lymphoid cellularity, and mitogen responses, but a potentially important immunologic deficit was found in these mice: 250 R caused a 70% loss of scid macrophages and dendritic cells which persisted at least 5 months. By contrast, when scid mice were injected i.p. with allogeneic bone marrow within the first 24 h after birth, rapid and complete reconstitution of both T and B cell lineages was achieved, and the animals had APC that were both donor and host in origin. Considering the extent and duration of engraftment (43 wk) by allogeneic cells in neonatally transplanted scid mice, it was anticipated that their bone marrow would be chimeric. However, the bone marrow contained very few donor-derived cells, suggesting that lymphopoiesis may be taking place in other organs in these chimeras.     

Linomide administration following bone marrow transplantation in mice

The effect of linomide, an immunomodulatory drug, on natural killer (NK) cells and T cell-dependent immune responses following syngeneic or allogeneic bone marrow transplantation (BMT) was investigated in BALB/c mice inoculated with B-cell leukemia (BCL1). Linomide given in the drinking water had no impact on graft survival or graft versus leukemia (GVL) effects. Although linomide regulates anti-self reactivity in mice with experimental and spontaneous autoimmune disorders, the anti-tumor effects induced by allogeneic donor lymphocytes were not affected. This indicates that different mechanisms regulate anti-self and anti-leukemia effects. Alternatively, linomide might affect the homing of self-reactive lymphocytes to specific target organs in autoimmune disorders, although the homing process may not be relevant to the control of leukemia by alloreactive lymphocytes.     

Hepatic ischemia/reperfusion injury involves functional TLR4 signaling in nonparenchymal cells

Endogenous ligands from damaged cells, so-called damage-associated molecular pattern molecules, can activate innate immunity via TLR4 signaling. Hepatic warm ischemia and reperfusion (I/R) injury and inflammation is largely TLR4 dependent. We produced TLR4 chimeric mice to assess whether the TLR4-dependent injury required TLR4 expression on liver parenchymal or nonparenchymal cells. Chimeric mice were produced by adoptive transfer of donor bone marrow cells into irradiated recipient animals using reciprocal combinations of TLR4 wild-type (WT; C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mouse bone marrow. Wild-type chimeric mice bearing TLR4 mutant hemopoietic cells and TLR4 mutant mice transplanted with their own bone marrow-derived cells were protected from hepatic I/R and exhibited decreased JNK and NF-kappaB activation compared with WT chimeric mice transplanted with their own bone marrow. In contrast, TLR4 mutant mice transplanted with TLR4 WT bone marrow were not protected from liver I/R and demonstrated pronounced increases in JNK and NF-kappaB activation when compared with autochthonous transplanted mutant mice. In addition, depletion of phagocytes taking up gadolinium chloride failed to provide any additional protection to TLR4 mutant mice, but substantially reduced damage in WT mice after hepatic I/R. Together, these results demonstrate that TLR4 engagement on actively phagocytic nonparenchymal cells such as Kupffer cells is required for warm I/R-induced injury and inflammation in the liver.     

Functional characteristics of a Ficoll-separated mouse bone marrow cell population involved in skin allograft prolongation

Treatment of recipient mice with donor bone marrow cells and anti-lymphocyte serum (ALS) results in extensive skin graft prolongation beyond that achieved in animals given only ALS. In this study, B6AF1 recipient mice were grafted with C3H/He skin on day 0, treated with ALS on days -1 and +2 and infused on day +7 with donor strain (C3H/He) bone marrow cells. Extensive graft prolongation was achieved either with 25 X 10(6) whole bone marrow cells or with 1 X 10(6) lymphoid-like cells derived from donor marrow that sediment at a rate of 3 mm/hr in a 2 to 4% Ficoll gradient at unit gravity. These allograft-prolonging lymphoid-like cells appear not to be CFUs cells, have suppressive activity in in vitro MLC assays, and contain both nylon wool adherent and non-adherent cells. These studies thus show that allograft promoting cells can be isolated from bone marrow utilizing Ficoll gradients. Functional studies suggest that 3 mm/hr sedimenting donor bone marrow suppressor cells may be involved in the induction of allograft prolongation.     

Alkaline and acid phosphatase activity in murine femoral bone marrow following X-irradiation, or X-irradiation and repopulation with syngenic or allogeneic bone marrow or marrow stroma cells

The activity of alkaline and acid phosphatases in the bone marrow from the femoral cavity was investigated in the following groups of mice: (1) normal (non-irradiated); (2) irradiated with 600 R; (3) irradiated and repopulated with syngeneic bone marrow; (4) irradiated and repopulated with syngeneic marrow stroma; (5) non-irradiated, infused with allogeneic bone marrow (host versus graft reaction, HvG); (6) irradiated and repopulated with allogeneic bone marrow (graft versus host reaction, GvH). In addition, the activity of alkaline and acid phosphatases was examined in bone marrow stromal cultures. In irradiated animals the activity of both enzymes was lower than in non-irradiated ones, repopulation with syngeneic bone marrow restoring it to normal. Repopulation with allogeneic marrow (GvH) resulted in a very deep reduction of alkaline, but not acid, phosphatase. It is postulated that the decrease in bone marrow alkaline phosphatase activity can be a sensitive test for the early GvH reaction, preceding such parameters as splenomegaly. Marrow stroma cultured in vitro also showed very low alkaline phosphatase activity.     

A model of Ph' positive chronic myeloid leukemia-blast crisis cell line growth in immunodeficient SCID mice.

A human Philadelphia-chromosome positive chronic myeloid leukemia-blast crisis (CML-BC) cell line BV173 proliferated in the hematopoietic tissues, infiltrated various organs and caused the death of immunodeficient SCID mice. Leukemia spreading was assessed with diminished number of bone marrow cells and caused splenomegaly. The leukemic colonies grew from single cell suspension of bone marrow, spleen and peripheral blood. Bcr-abl m-RNA was detectable in bone marrow, spleen, peripheral blood, liver, lungs and brain. Dying mice demonstrated severely hypoplastic bone marrow, splenomegaly and massive metastases in the liver and kidneys. The survival time of animals was dependent on the number of inoculated leukemia cells.     

Influence of a sublethal irradiation on the immune response of the mouse to sheep erythrocytes]

In the CBA mice, the immunological response of the spleen cells (RFC and PFC direct and indirect) against the sheep erythrocytes is highly depressed by a 400 R dose of X rays. The recovery is not complete at the 30th day after irradiation. The response of the bone marrow cells either irradiated or unirradiated to the antigenic stimulation is very low.     

Hematopoietic precursor cells in radiation chimeras restored by bone marrow from thymectomized mice]

Radioprotective capacity of bone marrow CFUs of adult thymectomized mice was studied. Lethally irradiated mice were inoculated with bone marrow of mice thymectomized 8-11 months before. The colony forming capacity and proliferative rate of CFUs were studied 1-7.5 months after obtaining the radiation chimeras. It has been shown that proliferative capacity of bone marrow of adult thymectomized mice was reduced in comparison with that of normal animals. It is related to the decrease (4-fold) of the proliferative rate of bone marrow of thymectomized mice which was inoculated into lethally irradiated recipients 1 month before. We also found that the content of CFUs in bone of those chimeras was reduced later--after 7.5 months. In this period (1-7.5 months) the cellularity of bone marrow did not change.     

脾加骨髓细胞移植治疗小鼠大肠癌的实验研究

目的:探讨半相合脾加骨髓细胞移植治疗小鼠大肠癌的效果及其对嵌合体水平和移植物抗宿主病(GVHD)的影响。方法:以接种CT26大肠癌细胞的BALB/c×C57BL/6杂交Fl代雌性小鼠为受鼠,以健康雌性Fl、雄性C57BL/6、雄性C3H小鼠为MHC全相合、半相合、不相合供鼠,观察移植后的抑瘤情况;另设5只化疗联合半相合脾加骨髓细胞移植的小鼠为监测组,用来作嵌合体的分析,观察各组GVHD的情况。结果:经化疗预处理的脾加骨髓细胞移植组小鼠肿瘤明显缩小,与单纯化疗未进行移植组比较,差异具有统计学意义(P0.05);化疗联合半相合脾加骨髓细胞移植的小鼠于移植3周后达完全供者植入,于移植后第10天左右出现纳差、倦怠、步态不稳、脱毛、腹泻、体重明显下降等GVHD的症状。结论:化疗预处理联合脾加骨髓细胞移植能对CT26大肠癌细胞产生GVT效应,并伴随着GVHD及嵌合率的变化。