Stem cell (CFU-s) proliferation in sublethally irradiated mice

Abstract. The proliferation rate of haemopoietic stem cells (CFU-s) was followed after sublethal total body irradiation with 1.5 Gy. The [³H]-thymidine suicide technique was used to measure the CFU-s proliferation rate. The measurements extended from 10 min after irradiation up to 21 days. The CFU-s did not enter the DNA synthesis period (S-phase) shortly after irradiation, as had been previously suggested, but did so only with a delay of 14–16 hr. A large scatter of results was explained by an oscillatory pattern in CFU-s proliferation. The CFU-s prepared for cell division in synchronized waves, with a period of 20–22 hr.     

Cellular aspects of tolerance. I. Parameters of tolerance induction in T cells of spleen and thymus

Unresponsiveness of T cells in thymus and spleen of tolerant animals was determined by reconstitution of lethally irradiated recipients. The degree of responsiveness of these animals was assessed by antigen elimination and two types of plaque assays (liquid and agar) with different sensitivity. Unresponsiveness occurred more rapidly in T spleen cells than in thymus cells. Unresponsiveness of T cells could be induced in the spleens of thymectomized animals and in T cell repopulated thymectomized lethally irradiated recipients. Induction of unresponsiveness did not depend on proliferating bone marrow cells or on accessory cells.     

Survival and Repopulation of Irradiated 'Pre-Cfu-C' In Mice

ABSTRACT Supernatants of murine bone-marrow cultures contain a colony-promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi-solid agar cultures in the presence of colony-stimulating factor (CSF). Incubation of bone-marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU-c) and the CPF-responsive cells may be younger than the CFU-c. We have investigated the radiosensitivity and the pattern of the recovery after irradiation of CPF-responsive cells. We found that the radiosensitivity of CPF-responsive cells was significantly lower than those of CFU-c. burst-forming units-erythroid (BFU-e) and pluripotent stem cells in vivo (CFU-s) and in vitro (CFU-mix). the CPF-responsive cells remained subnormal even at 28 days after irradiation of the mice, a time when the CFU-s and CFU-c had recovered completely. Therefore the CPF-responsive cells may constitute a separate compartment, namely ‘pre-CFU-c’, in the maturation sequence of granulopoiesis, and this maturation of the ‘pre-CFU-c’ to CFU-c seems to be highly stimulated after irradiation to counterbalance the influx from CFU-s.     

Non-circadian rhythm in proliferation of haematopoietic stem cells

The proportion of haematopoietic stem cells (CFU-s) engaged in DNA synthesis was determined by means of the [3H]-thymidine [( 3H]TdR) suicide technique during recovery of bone marrow from the damage caused by a sublethal total body irradiation. In contrast with previous reports the [3H]TdR suicide rate was not permanently increased. It was observed that CFU-s passed through S phase in synchronous waves, following a dose of irradiation of 1.5 Gy. After a dose of 2.6 Gy, there was only one initial wave of increased CFU-s sensitivity to the action of [3H]TdR. Following the depression occurring 26 hr after the irradiation with 2.6 Gy, the proportion of CFU-s killed by the [3H]TdR was permanently increased until 5-6 days after irradiation. Thereafter large differences in the [3H]TdR suicide data were observed among individual mice. Evidence was obtained that individual mice, which had been irradiated by a dose of 2.6 Gy 8-9 days before, had identical values of the CFU-s [3H]TdR suicide rate in the bone marrow from different bones of the lower extremities. The recurrence of the synchronous waves in CFU-s passage through the cell cycle was recorded when the CFU-s population regenerated to only about 10% of its normal value. These waves were obviously not related to a particular time of the day and, consequently, they did not represent the circadian rhythm. It is concluded that the synchronous waves in which CFU-s proliferation occurred reflected the action of the control mechanism on CFU-s proliferation. This mechanism should be endowed with an important systemic component besides locally operating factors.     

Survival and repopulation of irradiated 'pre-CFU-c' in mice

Supernatants of murine bone-marrow cultures contain a colony-promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi-solid agar cultures in the presence of colony-stimulating factor (CSF). Incubation of bone-marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU-c) and the CPF-responsive cells may be younger than the CFU-c. We have investigated the radiosensitivity and the pattern of the recovery after irradiation of CPF-responsive cells. We found that the radiosensitivity of CPF-responsive cells was significantly lower than those of CFU-c, burst-forming units-erythroid (BFU-e) and pluripotent stem cells in vivo (CFU-s) and in vitro (CFU-mix). The CPF-responsive cells remained subnormal even at 28 days after irradiation of the mice, a time when the CFU-s and CFU-c had recovered completely. Therefore the CPF-responsive cells may constitute a separate compartment, namely 'pre-CFU-c', in the maturation sequence of granulopoiesis, and this maturation of the 'pre-CFU-c' to CFU-c seems to be highly stimulated after irradiation to counterbalance the influx from CFU-s.     

Regulation of haemopoietic stem-cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Sl^j allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone-marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU-s) proliferation in Sl^j/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU-e and CFU-G/M) kinetics after TBI and +/+ bone-marrow transplantation in Sl^j/+ and +/+ mice. the Sl^j/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Sl^j/+ spleen colonies contained the same number of BFU-E and CFU-G/M as colonies from +/+ spleens, while their CFU-s content was increased. On day 10 post-transplantation, the macroscopic ‘missing’ colonies could be detected at the microscopic level. These small colonies contained far fewer CFU-s than the macroscopic detectable colonies. Analysis of CFU-s proliferation-inducing activities in control and post-LPS sera revealed that Sl^j/+ mice are normal in their ability to produce and to respond to humoral stem-cell regulators. We postulate that Sl^j/+ mice have a normal number of splenic stromal ‘niches’ for colony formation. However, 35% of these niches is defective in its proliferative support.     

Characteristics of the Cfu-S Population In Mice Carrying the SlJ Allele

Abstract Abstract. A tentative characterization of haemopoietic stem cells with respect to their organ distribution, seeding fraction and colony formation in the spleen, radiosen-sitivity and humoral regulation was attempted in mice heterozygous for the mutant allele Sl^J and in their normal littermates. Sl^J/+ mice were characterized by a deficient CFU-s content of the blood and spleen and had slightly lower femoral CFU-s numbers. This CFU-s distribution could not be explained by differences in seeding efficiency ‘f’ between CFU-s of Sl^J/+ and +/+ origin in lethally irradiated recipients used in the CFU-s assay. the seeding fraction of CFU-s of +/+ origin did not differ in +/+ and Sl^J/+ recipients. However, in irradiated SI^J/+ recipient mice a 30% decrease was observed in the number of the colonies derived from splenic and femoral CFU-s of both +/+ and Sl^J/+ origin. the serum level of SHSF (splenic haemopoiesis stimulating factor) was decreased in Sl^J/+ mice, but significantly increased in Sl/Sl^d mice, as compared to their respective normal +/+ littermates. Endogenous colony formation in Sl^J/+ spleens was deficient in comparison to that observed in +/+ spleens, and distinct sex differences were observed. However, mutant and normal CFU-s from spleen and bone marrow had a similar survival following in-vitro y irradiation. Femurs and spleens of both Sl^J/+ and +/+ origin were implanted into both Sl^J/+ and +/+ hosts. Six weeks later the Sl^J/+ grafts contained less CFU-s than the +/+ grafts. These data show that the splenic stroma of Sl^J/+ mice is not defective in its capacity to lodge injected CFU-s but is deficient in its ability to maintain CFU-s under ‘steady-state’ conditions and stimulate their colony formation in a ‘perturbed state’. Some of the characteristics of Sl^J/+ mice segregate them from Sl/Sl^d mice, i.e. a deficient splenic CFU-s content, normal seeding fractions ‘f’ of CFU-s from spleen and bone marrow in the presence of an almost compensated anemia, and decreased serum levels of SHSF. the study of the Sl^J trait may be a useful extension of the current Sl/Sl^d model for exploration of hereditary defects in haematopoietic stroma.     

Non-Circadian Rhythm In Proliferation of Haematopoietic Stem Cells

Abstract. The proportion of haematopoietic stem cells (CFU-s) engaged in DNA synthesis was determined by means of the [³H]-thymidine ([³H]TdR) suicide technique during recovery of bone marrow from the damage caused by a sublethal total body irradiation. In contrast with previous reports the [³H]TdR suicide rate was not permanently increased. It was observed that CFU-s passed through S phase in synchronous waves, following a dose of irradiation of 1.5 Gy. After a dose of 2.6 Gy, there was only one initial wave of increased CFU-s sensitivity to the action of [³H]TdR. Following the depression occurring 26 hr after the irradiation with 2.6 Gy, the proportion of CFU-s killed by the [³H]TdR was permanently increased until 5-6 days after irradiation. Thereafter large differences in the [³H]TdR suicide data were observed among individual mice. Evidence was obtained that individual mice, which had been irradiated by a dose of 2.6 Gy 8-9 days before, had identical values of the CFU-s [³H]TdR suicide rate in the bone marrow from different bones of the lower extremities. the recurrence of the synchronous waves in CFU-s passage through the cell cycle was recorded when the CFU-s population regenerated to only about 10% of its normal value. These waves were obviously not related to a particular time of the day and, consequently, they did not represent the circadian rhythm. It is concluded that the synchronous waves in which CFU-s proliferation occurred reflected the action of the control mechanism on CFU-s proliferation. This mechanism should be endowed with an important systemic component besides locally operating factors.     

A Study of the Regenerative Potential of Bone Marrow Cells of Donor Mice that Carry the <Emphasis Type="Italic">egfp</Emphasis> Gene in Irradiated Mice

A study of the regenerative potential of bone marrow cells of donor mice that express the enhanced green fluorescent protein was conducted in mice irradiated at a dose of 7 Gy. Expression of this protein allowed us to carry out monitoring of the presence of donor cells in recipient blood over the entire lifespan of the recipient. The lifespan of young recipients increased by 93% after transplantation; for old recipients it increased by 15%. Total acceptance of the bone marrow, spleen, thymus, and blood of the recipient with donor bone marrow cells was demonstrated over the entire life of the recipient. Only the donor colonies were detected with the studied irradiation dose and number of transplanted cells (11.7 ± 0.4) · 10⁶ on the spleen surface. The percentage of bone marrow and spleen cells that expressed the CD117 and CD34 stem cell markers in the recipient mice was above the control level for a long period of time after the irradiation. More than half of the cells with CD117, CD34, CD90.2, and CD45R/B220 phenotypes in the studied organs were donor cells. Further detailed study of the peculiarities of the engraftment of bone marrow cells, both without preliminary treatment of recipients and after the effects of extreme factors, will allow improvement of the methods of cell therapy.     

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.     

T cell differentiation in lethally irradiated and reconstituted mice: stem cell influx demonstrated with immunoperoxidase technique.

Using both an anti-stem cell serum and an anti-T cell serum the influx of stem cells in mouse thymus and spleen after lethal irradiation and reconstitution was determined by immunoperoxidase staining. In both organs a rapid influx was observed reaching a maximum on Day 5 after irradiation and cell transfer. Thereafter a decline of stem cells took place while the number of T cells in the thymus increased gradually, reaching a maximum on Day 12. T cells could only be detected in the spleen after 3 weeks.     

In vivo studies on the regeneration kinetics of enriched populations of haemopoietic spleen colony-forming cells from normal bone marrow

Functional properties of mouse haemopoietic spleen colony-forming cells, enriched 40- to 80-fold, from normal bone marrow were studied. It was found that: (1) the number of partially purified CFU-s (colony forming unit-spleen) required to rescue lethally irradiated mice was similar to the number of normal unfractionated bone marrow CFU-s giving the same level of protection; (2) the homing of partially purified CFU-s was similar to that of CFU-s from unfractionated bone marrow; (3) the regeneration of CFU-s in spleen was similar for enriched and unfractionated cell populations between 4 and 11 days after transplantation. In contrast, the rate of regeneration of CFU-s in femur was slower with enriched progenitor cells than with unfractionated bone marrow. The growth rate in femur, however, could be restored to normal by injecting freshly isolated syngeneic thymocytes with the enriched CFU-s population. The results indicate that the partially purified CFU-s are by themselves functionally normal and show that the rate of CFU-s repopulation in bone marrow can be affected by cell types other than spleen colony-forming cells.     

Cells Forming Spleen Colonies At 7 Or 11 Days After Injection Have Different Proliferation Rates

Abstract In the early periods (7–9 days) after haemopoietic cell injection, colonies produced by CFU-s and by their progeny are identified in the spleen, while at later periods (11 days after injection) only spleen nodules produced by CFU-s persist. the increase in the suicide values of CFU-s after sublethal (2 Gy) irradiation of mice is associated with a higher proliferation rate of precursors of transitory spleen colonies, but not of CFU-s, as measured by different suicide techniques. During the log-phase of cell growth in a lethally irradiated recipient, the injected CFU-s and CFU-tr proliferate at a higher rate. Active proliferation of CFU-s and CFU-tr has been demonstrated in long-term bone-marrow cultures by the hydroxyurea in-vitro suicide assay. CFU-tr may be the cause of artifactual effects during measurement of haemopoietic stem-cell cycling by CFU-s suicide methods.     

Thymus dependency of cells involved in transfer of delayed hypersensitivity to listeria monocytogenes in mice

Delayed-type hypersensitivity (DH) to Listeria antigens was induced in inbred C3Hf/Umc mice by intravenous injection of a sublethal dose of viable Listeria monocytogenes. Bone marrow, spleen, and lymph node cells from the immune mice were capable of passive transfer of DH to syngeneic neonatally thymectomized or lethally (900 R) irradiated recipients. Immune thymus cells as well as immune serum were ineffective in transferring DH to irradiated animals. In vitro treatment with antitheta isoantibody (anti-θ) and complement abolished the capacity of spleen and bone marrow cells from immune donors to transfer DH to irradiated hosts, indicating the thymus dependency of this cell population. The results with bone marrow indicate the existence of a small, but biologically significant, thymus-dependent population in this tissue.     

Transfer of immunity by transfer of bone marrow cells: T-cell dependency.

Thymectomized, lethally irradiated mice reconstituted with normal bone marrow cells succumbed when challenged ip with rat Yoshida ascites sarcoma (YAS) cells 40 days after irradiation and reconstitution. In contrast, thymectomized irradiated mice reconstituted with bone marrow cells from YAS-immune donors rejected the subsequent tumor challenge. Pretreatment of the bone marrow cells from immune donors with anti-Thy 1.2 antiserum and complement completely abolished the transfer of anti-YAS resistance.Bone marrow cells from donors thymectomized 2 months before immunization enabled almost all recipients to reject YAS, but bone marrow cells from donors thymectomized 8 months before immunization protected only 50% of the recipients. Further analysis showed that mice thymectomized 8 months before immunization failed to generate anti-YAS antibody response, whereas the antibody response of mice thymectomized 2 months before immunization did not differ from that of non-thymectomized age-matched control mice. The data suggest that the immune reaction of mice against xenogeneic YAS requires long-lived T₂ lymphocytes.     

Inhibition of the endocrine function of the rat thymus by x-irradiation

Whole-body X-irradiation of rats caused inhibition of endocrine function of thymus. The effect was a function of radiation dose and time after irradiation. 72 h following irradiation with doses of 6 and 8 Gy the thymus hormone content of blood serum fell down the level registered in the thymectomized animals. Cellularity of the thymus and spleen concurrently decreased. The kinetics of spontaneous chemiluminescence of blood serum, thymus and spleen cells characterized the hypersecretion of glucocorticoids in response to radiation activation of lipid peroxidation in radiosensitive rat organs.     

CD4+ T cells in aged or thymectomized recipients of allogeneic stem cell transplantations

Background

CD4+CD25highFOXP3+ regulatory T (Treg) cells, which include thymus-derived and peripherally induced cells, play a central role in immune regulation, and are therefore crucial to prevent graft-versus-host disease (GVHD). The increasing use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for elderly patients with thymus regression, and our case of allo-HSCT shortly after total thymectomy, raised questions about the activity of thymus-derived Treg cells and peripherally induced Treg cells, which are otherwise indistinguishable.

Results

We found that despite pre-transplant thymectomy or older age, both naïve and effector Treg cells, as well as naïve and effector conventional T cells, proliferated in allo-HSCT recipients. Higher proportions of total Treg cells 1 month post allo-HSCT, and naïve Treg cells 1 year post allo-HSCT, appeared in patients achieving complete chimera without developing significant chronic GVHD, including our thymectomized patient, compared with patients who developed chronic GVHD.

Conclusions

Treg cells that modulate human allogeneic immunity may arise peripherally as well as in the thymus of allo-HSCT recipients.     

Age-related decrease in mouse T cell progenitors.

Mice were given a lethal dose of whole-body gamma-radiation and injected with a 10(5) or 10(6) marrow cells from 10- to 143-week-old syngeneic donors. Nine days later, colony-forming units (CFU) were counted in the spleens of mice given 10(5) cells, and 15 to 21 days after irradiation thymus weights and in some experiments 3H-thymidine uptake or total thymic cellularity were determined in the recipients of 10(6) cells. It was found that in the majority of mouse strains studied there were no significant changes with age in marrow CFU. In contrast, thymic regeneration was significantly impaired when the recipients received marrow cells from donors 100 weeks of age or older. These observations and results obtained in dose-response and time-course studies are best explained by an age-related decrease in marrow T cell progenitors; however, certain findings suggest that in addition the proliferative capacity of these stem cells may at times be moderately impaired.     

Restoration of the Immune Response to Sheep Erythrocytes by a Serum Factor

THE immune response of CBA mice to sheep erythrocytes (SRBC) is known to be thymus-dependent because strain members thymectomized during the first few hours of life exhibit a marked inability to respond to this antigen^1,2. Experiments with isoantisera suggested that a cell to cell interaction is involved in this response. Thymus cells per se do not develop into haemolytic plaque-forming cells, but in some, so far obscure, way they cause cells of bone marrow origin to become producers of haemolytic plaques^2,3. A study of spleen cells from neonatally thymectomized (NNT) mice in a tissue culture system indicated that the decreased responsiveness to SRBC is also expressed in vitro. In that case 15×10⁶ NNT spleen cells produced only 500 haemolytic plaques when assayed on day 4 of culture. But when 15×10⁶ thymus cells were added to identical cultures of NNT spleen cells at inception, the number of haemolytic plaque forming cells increased to 2,300 (ref. 4). When an equivalent number of thymus cells alone were incubated with SRBC there was no response.     

Production of presumptive humoral haematopoietic regulators in canine cyclic haematopoiesis

Abstract. Conditioned media (CM) were prepared according to previously published techniques from the bone marrow of dogs with cyclic haematopoiesis (CH). CM prepared from day 9 marrows inhibited mouse bone marrow CFU-s proliferation rate while CM from day 10 marrows were stimulatory and also contained an erythroid stimulating factor which appeared to be erythropoietin. In addition a highly significant trend from CM containing CFU-s inhibitory materials to media with CFU-s stimulatory activity was observed through cycles day 1 to 8. These studies further support the concept that CH is due to a defect in factors controlling stem cell proliferation and suggest that a major event occurs in CH dog marrow on days 9 and/or 10 of the cycle. Bone marrow transplantation studies (Dale & Graw, 1974; Weiden et al., 1974; Jones et al., 1975b) have indicated that canine cyclic haematopoiesis (CH) is probably due to a disorder in the multipotential stem cells. Morphological evidence (Scott et al., 1973) and the almost synchronous cycling of CFU-e, CFU-c and diffusion chamber progenitor cells (DCPC) (DUM et al., 1977, 1978a, b) lend support to such a theory. However, efforts to identify the mechanisms controlliig multipotential stem cell proliferation in dogs have been handicapped by the lack of suitable techniques to study these cells in the canine. Recently, Wright and co-workers (Wright & Lord, 1978, 1979; Wright et al., 1979; Lord et al., 1979), on the basis of previous observations (Frindel et al., 1976; Frindel & Guigon, 1977), described the preparation of species non-specific, bone marrow conditioned media (CM) which are capable of influencing the proliferation rate of murine colony forming units-spleen (CFU-s). The studies now reported were designed to determine if CM prepared from canine CH marrow would influence the proliferation rate of murine bone marrow CFU-s. The results indicate that a major event, possibly related to the in vivo control of stem cell proliferation in dogs with CH, occurs on days 9–10 of the cycle; day 1 being the first day when the peripheral blood neutrophil count falls below-1600 mm³.