The onset of hemoglobin synthesis in spleens of irradiated mice after bone marrow transplantation.

Messenger RNA (mRNA) for globin was isolated from spleens of irradiated mice in which erythroid differentiation was induced by a bone marrow graft. The globin mRNA was isolated either by means of sucrose gradients of reticulocyte polysomal RNA or by affinity chromatography of total spleen RNA on poly (U)-sepharose. The globin mRNA was tested in a wheat embryo cell-free system. The appearance of mRNA in the spleen erythroid colonies was correlated with other parameters of erythroid differentiation such as globin synthesis, activity of delta-aminolevulinic acid synthetase and iron uptake. Poly(A) containing mRNA did appear already on the 3rd day after grafting. However, significant translational activity of globin mRNA could be demonstrated only one day later together with the increase in globin synthesis and delta-aminolevulinic acid synthetase and enhanced iron uptake. In the second part of this study mouse spleen cells rich in erythroid elements were incubated with a specific heme synthesis inhibitor (isonicotinic acid hydrazide, INH) and the synthesis of 9 S RNA was estimated. It was found that a 40-minute incubation with INH reduced uridine incorporation into 9 S RNA fraction by about 40%.     

Inactivation of stem cells by allogenic lymphocytes: competition between T1- and T2-subpopulations]

Transplantation of the bone marrow cells with allogeneic T-lymphocytes to the irradiated hosts was accompanied by inactivation of the stem elements of the graft. The lymph node cells of T-mice (those deprived of B cells) were more active than the spleen cells of these mice. The stem cells inactivation was weakly expressed or absent in case of a combined acti-n of T-cells from the lymph nodes and the spleen.     

Hemopoietic colonies on the chorioallantoic membrane of the chick embryo: Induction by embryonic,adherent, non-hemopoietic spleen cells

Granulocytic and erythrocytic colonies developed on the chick embryo chorioallantoic membrane (CAM) following the inoculation of chick embryo spleen cells. Dose response and kinetic experiments showed that the colonies were derived from cell aggregates present in the inoculum. Dissociation and reaggregation studies of the CAM colony-inducing cells (CAM-CIC) indicated that these cells must be present as aggregates in order to form colonies. Results from the morphology and cell marker experiments suggested that the colony-inducing aggregates (CAM-CIA) attract and support the differentiation of primitive host hemopoietic cells. The physical characteristics of the CAM-CIC, which are different from those of the hemopoietic progenitor cells, indicated that they represent a stromal cell population of the chick embryo spleen. Further evidence supporting this notion was provided by the radiation studies which showed that the colony-inducing ability of the CAM-CIC is radioresistant. The above characteristics of the CAM-CIC strongly suggest that they represent the stromal cells of the chick embryo spleen which influence hemopoiesis.     

Production of cytokines after lipopolysaccharide stimulation of murine spleen cells during lymphoma development in AKR mice

Summary Injection of syngeneic lymphoma cells in AKR mice resulted in an important increase of splenic natural killer (NK) activity in the early days following the graft. Modifications of the production of different types of cytokine: interferon, interleukins 1 and 2, tumor necrosis factor (IFN, IL-1, IL-2, TNF), involved in the regulation of NK activity, were investigated in short-term cultures of total, adherent and non-adherent fractionated spleen cells, using lipopolysaccharide as the triggering or amplifying agent.Upon stimulation with lipopolysaccharide, splenocytes from lymphoma-grafted mice released a large amount of interferon as compared to controls with a maximum level 1 day after the graft. Equal amounts of IFN- and IFN-/ were detected. Treatment of spleen cells prior to culture with anti-(asialo-GM1) or anti-(Thy-1.1) antibodies reduced interferon production by 80% and 50% respectively. This finding indicates that (a) the IFN- is produced by Thy-1-positive cells and (b) the production of IFN- by these cells is at least partially under the control of asialo-GM1-positive cells. We also showed that non-adherent fractionated spleen cells from lymphoma-grafted mice produced IL-1 and IL-2. IL-1 was released by asialo-GM1-positive cells and IL-2 by Thy-1-positive cells. Adherent cells released only IL-1. In contrast, total cells released smaller amounts of IL-1 and IL-2, suggesting a reciprocal inhibition between subpopulations of non-adherent and adherent cells. A high level of TNF production by adherent cells was observed only 4 days after the graft. These results indicate that graft of lymphoma cells entails important modifications of spleen cell populations releasing different types of cytokines implicated in NK activation.     

Splenomegaly reaction of the chick embryo following chorio-allantoid graft of chicken-spleen fragments]

The spleen enhancement reaction of the chick embryo, following the insertion (grafting or injection procedure) of homologous spleen cells is one of the results of the graft-versus-host reaction (G.V.H. reaction). Irrespective of the usual kinds of G.V.H. reaction measured, it has been proved that the relation between the number of immuno competent cells and the reaction intensity is linear. Our study shows that the relation is not the same when the chorio-allantois membrane grafting procedure is used instead of injection into the veins. However, two facts remain unchanged 1) the minimal amount of spleen cells sufficient to provoke a spleen enhancement is low, 2) there is a link between the number of homologous spleen cells and the rate of spleen enhancement, but in this case it was not shown to be linear. In the light of this, the role played by the chorio-allantois membrane is being debated.     

Induction of tolerance to parental marrow grafts in F1 hybrid mice. Evidence for recognition of self-antigens

Lethally irradiated (C57BL/6xC3H)F1 mice are able to acutely reject parental C57BL/6 but not C3H marrow grafts, a phenomenon called hybrid resistance (HR). In attempts to inactivate this rejection mechanism we found that parental spleen cells activated with LPS are very potent in inducing tolerance to a subsequent C57BL/6 marrow graft. Tolerance is likely due to elimination of effector cells responsible for graft rejection as adoptive transfer of spleen cells from normal into tolerized mice reconstitutes responsiveness. Evidence is presented that the Ag on LPS-activated spleen cells responsible for induction of unresponsiveness are expressed on both C57BL/6 and (C57BL/6xC3H)F1 cells. This suggests that the HR effector cells recognize autoantigens. In support of this, induction of tolerance to C57BL/6 parental marrow grafts leads to a concomitant dramatic increase in endogenous CFU-spleen after a dose of gamma-irradiation. Moreover, elimination of the cells responsible for HR by injection of anti-ASGM1 antibody results in a similar increase of endogenous CFU-spleen after irradiation. It is concluded that HR is a reflection of autoimmunity, able to limit the proliferation of syngeneic marrow stem cells.     

In situ effector pathways of allograft destruction. 2. Generation of the "humoral" response in the graft and the graft recipient

The frequency of both immunoglobulin (Ig)-synthesizing and Ig-secreting B cells have been analyzed in DA-to-WF rat renal allografts (and in control WF-to-WF autografts). We have correlated the in situ B-cell responses with corresponding events in the central lymphatic system of the recipient. Intracellular IgM- and IgG-containing plasma cells appeared in an allograft (but not in an autograft) very shortly after the transplantation. The numbers of both cell types in situ was approximately equal, the highest numbers of each being found on Day 4 after transplantation. A similar early response was observed in the recipient's spleen, however, very few Ig-synthesizing cells were seen in the blood. Only a fraction of the Ig-synthesizing cells in the allograft were involved in immunoglobulin secretion. Thus, the recovery of IgG- and IgM-secreting cells from an allograft was 10 and 2% of intracellular IgG- and IgM-containing cells, respectively. It appears, therefore, that allograft-infiltrating Ig-synthesizing B cells either die or migrate elsewhere before secreting immunoglobulin. The B-cell response in the graft occurs very early and is disproportionally high when the very low frequency of B lymphocytes in the allograft is considered. The data provide no evidence for inflammatory B cells being an integral part of graft rejection. Indeed, the possibility remains that the inflammatory B-cell response observed during the rejection process represents a meaningless byproduct of the inflammatory response.     

Cell lineage analysis of neural induction: origins of cells forming the induced nervous system

Horseradish peroxidase (HRP) was used as an intracellular lineage tracer in two experiments designed to reveal the sites of origin of cells that formed the duplicate embryo which developed in relation to an organizer grafted in the ventral marginal zone (VMZ) of Xenopus laevis embryos. In the first experiment a dorsal blastoporal lip fully labeled with HRP was grafted in the VMZ of an unlabeled embryo at the beginning of gastrulation. This resulted in development of a second embryo in which labeled cells, of graft origin, formed the notochord, and parts of the somites, endoderm, and neural tube. The second experiment was designed to show the sites of origin of the host's cells that formed parts of the induced embryo. HRP was injected into individual blastomeres in a series of Xenopus embryos at the 32-cell stage and each embryo received an unlabeled organizer graft in the VMZ at the beginning of gastrulation. In these embryos the lineages that contributed to the host's primary neural tube did not contribute any cells to the induced neural tube. All the cells in the induced neural tube which originated from the host were descendants of ventral blastomeres that did not contribute to the neural tube normally. This shows that the second neural tube is formed as a result of the action of the organizer on cells in its immediate vicinity which would not normally have entered neural pathways of differentiation.     

Production of an antiviral factor by murine spleen cells after treatment with Corynebacterium parvum.

We have previously shown that injection of Corynebacterium parvum (CP) in mice protected them against lethal encephalitis induced by herpes simplex virus, (HSV). It is shown here that spleen cells of CP-injected mice in vitro produce a factor capable of inhibiting the replication of HSV in mouse embryo fibroblasts (MEF). A similar activity was produced after in vitro exposure of spleen cells from untreated mice to CP. CP was only slightly mitogenic in contrast to phytohemagglutinin (PHA), concanavalin A (Con A), and bacterial lipopolysaccharide (LPS) which were strongly mitogenic but did not induce antiviral activity high enough to be detected in the HSV-MEF system. The activity produced by CP-treated spleen cells appeared to be interferon since it was trypsin sensitive and species specific and not virus specific, and since preincubation of the cells was required to demonstrate an antiviral effect. However, the identity of CP-induced interferon with any of the previously described subclasses of interferon remains to be determined.     

Immune reconstitution after anti-thymocyte globulin-conditioned hematopoietic cell transplantation

Background aimsAnti-thymocyte globulin (ATG) is being used increasingly to prevent graft-versus-host disease (GvHD); however, its impact on immune reconstitution is relatively unknown. We (i) studied immune reconstitution after ATG-conditioned hematopoietic cell transplantation (HCT), (ii) determined the factors influencing the reconstitution, and (iii) compared it with non-ATG-conditioned HCT.MethodsImmune cell subset counts were determined at 1–24 months post-transplant in 125 HCT recipients who received ATG during conditioning. Subset counts were also determined in 46 non-ATG-conditioned patients (similarly treated).Results(i) Reconstitution after ATG-conditioned HCT was fast for innate immune cells, intermediate for B cells and CD8 T cells, and very slow for CD4 T cells and invariant natural killer T (iNKT) (iNKT) cells. (ii) Faster reconstitution after ATG-conditioned HCT was associated with a higher number of cells of the same subset transferred with the graft in the case of memory B cells, naive CD4 T cells, naive CD8 T cells, iNKT cells and myeloid dendritic cells; lower recipient age in the case of naive CD4 T cells and naive CD8 T cells; cytomegalovirus recipient seropositivity in the case of memory/effector T cells; an absence of GvHD in the case of naive B cells; lower ATG serum levels in the case of most T-cell subsets, including iNKT cells; and higher ATG levels in the case of NK cells and B cells. (iii) Compared with non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT was slower for CD4 T cells, and faster for NK cells and B cells.ConclusionsATG worsens the reconstitution of CD4 T cells but improves the reconstitution of NK and B cells.     

Induction of graft versus leukemia effects by cell-mediated lymphokine-activated immunotherapy after syngeneic bone marrow transplantation in murine B cell leukemia

 The feasibility of inducing graft versus leukemia (GVL) effects with allogeneic T cells in recipients of autologous bone marrow transplantation (BMT) was studied in a murine model (BCL 1) of human B cell leukemia/lymphoma. Allogeneic cell therapy, induced by infusion with peripheral blood lymphocytes, a mixture of allogeneic spleen and lymph node cells and allogeneic activated cell therapy, induced by in vitro recombinant-interleukin-2(rIL-2)-activated allogeneic bone marrow cells in tumor-bearing mice, prevented disease development in adoptive BALB/c recipients. Concomitant in vivo activation of allogeneic lymphocytes with rIL-2 suppressed even more effectively the development of leukemia in secondary adoptive recipients of spleen cells obtained from treated mice. In contrast, in vivo administration of rIL-2 after syngeneic BMT, with or without equal numbers of syngeneic lymphocytes, led to disease development in secondary recipients. Our data suggest that effective cell therapy can be achieved after SBMT by allogeneic but not syngeneic lymphocytes and that anti-leukemic effects induced by allogeneic lymphocytes can be further enhanced by in vitro or in vivo activation of allogeneic effector cells with rIL-2. Therefore, cell therapy by allogeneic lymphocytes following autologous BMT could become an effective method for inducing GVL-like effects on minimal residual disease provided that graft versus host disease can be prevented or adequately controlled. Received: 14 May 1996 / Accepted: 6 August 1996     

On the behaviour of murine lymphocytes after in vitro treatment with acid mucoproteins from human serum.

Seromucoproteins from human serum were isolated by perchloric acid extraction followed by DEAE-Sephadex A-50 ion exchange chromatography. The in vitro pretreatment of spleen leukocytes with this fraction caused a dose-dependent inhibition of graft-versus-host reaction as well as an increase of their electrophoretic mobility, the viability being maintained. On contrary, the pretreatment of mice (prospective spleen cell donors of recipients of sheep red blood cells) with human seromucoproteins had no effect on the gvh-reaction as well as on the agglutinin formation to sheep red blood cells under the given conditions. It is supposed that the suppressive effect after in vitro pretreatment may be attributed to a coating effect of seromucoproteins. The fact that spleen cells pretreated in vitro with seromucoproteins are lysed in presence of complement and antiseromucoprotein antiserum supports our opinion. These findings as well as data from the literature support the hypothesis that local concentrated mucoproteins in the skin graft bed in cases of protractedly surviving skin grafts, in the placenta, and on neoplastic tissues can influence unspecifically the immune response. We hope that the understanding of this mechanism may open new possibilities in prolonging allograft survival time.     

Studies on the cytosolic DNA of chick embryo fibroblasts and its uptake by recipient cultured cells

Cytosol and extruded DNA complexes from cultured chick embryo fibroblast cells have been separated by agarose gel chromatography at intervals after pulse labelling with [3H]thymidine. The proportion of the various cytosol components changed markedly with time: there was a lag period of 3 hr before the major labelled (5 X 10(5) dalton) DNA complex appeared in the cytosol, and a further lag period of 5 hr before it was extruded from the cell. Cultured chick embryo fibroblast, and rat spleen, cells rapidly and very efficiently import their own or each others cytosolic DNA complexes into their respective cytosol fractions: the material recovered from the cytosol of recipient cells is characteristic of the presented material. Homologous cytosolic DNA complex presented to chick embryo fibroblast cells also becomes associated with the nucleus. The rat at which this occurs is comparable with the rate of incorporation of [3H]thymidine into nuclear DNA.     

Cell division in the ciliary ganglion of quail embryos in situ and after back-transplantation into the neural crest migration pathways of chick embryos

Embryonic 4- to 15-day-old quail ciliary ganglia (CG) were grafted into the neural crest migration pathway of 2-day-old chick embryos at the adrenomedullary level of the neural axis. This back-transplantation results in dispersion of cells of the implanted ganglion, their migration in the host embryo, and subsequent promotion of their differentiation into a variety of neural-crest-derived cell types including adrenergic cells of the sympathetic ganglia and adrenal medulla. These cells can be recognized in the host through the nuclear marker that they carry. Here, we have analyzed quantitatively the expansion of CG-derived cell population after the graft, and compared cell division in CG after back-transplantation and during normal in situ development over the same period of time. Tritiated-thymidine [( 3H]TdR) incorporation showed that grafted CG cells proliferated during their migration and, to a greater extent, after they had homed to the host structures. Furthermore, proliferative activity of quail cells in the graft was found to be significantly higher than the growth rate of the CG cells in situ during the same period of development. In the quail donor embryo, the birthdate of the CG neurons occurred early in development; from 6 days onward, only nonneuronal cells were still dividing. When back-transplanted, the 4- to 5-day-old CG provided numerous quail cells located in autonomic structures of the host embryo. However, this increase of the total quail cell population and of cell division was reduced when CG were taken from quail donors at progressively later developmental stages. Postmitotic neurons from mature CG were found not to survive under the graft conditions. It is proposed that back-transplantation of the CG stimulates cell division and modifies the developmental programme of still undifferentiated precursor cells which then can give rise to a variety of cell types belonging either to the glial or the autonomic nerve and paraganglionic cell phenotypes, to the exclusion of sensory neurons which never derive from CG grafts.     

In situ effector pathways of allograft destruction. 1. Generation of the "cellular" effector response in the graft and the graft recipient

Inflammatory leukocytes of DA-to-WF rat renal allografts displayed significant cytolytic activity to natural killer (NK) target cells on Day 2 after transplantation. The NK activity, which was associated with large granular lymphocytes in discontinuous Percoll gradients, peaked on Day 4 and disappeared rapidly thereafter. Coincident with the presence of NK activity in the graft, a decrease in NK activity in the recipient spleen was observed. Low NK activity was also recorded in WF-to-WF autografts. The cells displaying direct cytotoxic activity to donor (but not to recipient) strain peritoneal exudate target cells (PEC) were associated with the T suppressor/killer lymphocytes in affinity chromatography. They appeared in the graft between Days 2 and 4, peaked between Days 6 and 8 and disappeared slowly thereafter. In the spleen the cytotoxic T lymphocyte (CTL) activity appeared later and it reached a maximum between Days 16 and 20 before decreasing. In the blood distinct CTL activity was seen only from Days 16-20 onwards, after the graft had been rejected. No CTL activity was recorded in the graft, blood, or spleen of an autograft recipient. Addition of donor-directed post-transplantation antibody (antibody-dependent cellular cytotoxicity, ADCC) had a slight enhancing effect on the cytotoxic activity of inflammatory leukocytes up to Day 5. After this time, added antibody had a blocking effect on direct CTL activity. No ADCC activity was recorded in the inflammatory population of an autograft. On the contrary, high levels of ADCC activity to donor strain PEC were recorded in the spleens of both autograft and allograft recipients throughout the period of follow-up. The results demonstrate that at least three cellular effector pathways exist in an allograft: a strong natural killer cell component, a strong cytotoxic T lymphocyte component, and (possibly) a weak cell component participating in an ADCC type of cytotoxicity.     

Spleen enhancement reactions in chick embryo after homografting adult chicken spleen fragments: changes in spleen cell population in relation to rate of spleen enhancement]

A method using the comparison between the modifications of spleen weight and on spleen smears visible modifications of the distribution into categories of cells which are classified by the mean of their maturation degree, suggested that these both types of modifications are bound. Therefore, the classic cytological study seems to be a really good tool for analysing the effects of this kind of grafting on the haemopoietic organs, the weight modifications of which are difficult to measure. On the other hand, this method allowed us to see that the distribution of the cell population in the weakly enlarged spleens is very different from both distribution of the population in the strongly enlarged spleens and the controls ones ; this heterogeneity of the stimulating effect of the graft asked us the question whether this one could exerce its influence by the mean of two different ways, at least, or by only one.     

Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus

We examined the timing and mechanisms of mesodermal and neural determination in Cynops , using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops .     

Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.     

Developmental abnormalities of NT mouse embryos appear early after implantation

In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.     

A COMPARATIVE STUDY OF THE REPOPULATING POTENTIAL OF GRAFTS FROM VARIOUS HAEMOPOIETIC SOURCES: CFU REPOPULATION

The growth rate of the CFU populations in spleens and femora has been studied in irradiated mice injected with cell suspensions, containing equivalent number of CFU, from various sources. The doubling times are shown to be dependent upon the source of the cells. Grafts of bone marrow, spleen and foetal liver cells produced doubling times in the spleen of approximately 25, 19 and 16 hr respectively. Grafts of marrow-derived and spleen-derived spleen colony cells both gave rise to CFU doubling times lower than those of the corresponding primary grafts (approx. 33 and 26 hr respectively in the spleen). In the case of both bone marrow and spleen grafts the CFU population growth was shown to be independent of the size of the graft. A hypothesis is advanced which invokes at least a dual population of CFU, having different doubling times, different seeding capacities in the haemopoietic tissues following i.v. injection and present in different proportions in the various haemopoietic tissues.