Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation Ii. Physical Characteristics of Thymocyte Progenitor Cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H × AKR)F₁ bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, thymocyte progenitor cells show the same distribution as CFUs. the relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Effect of fractionated thymocytes from intact and irradiated mice on CFU recovery in the bone marrow after sublethal irradiation

In studying the influence of thymocytes fractionated by their size in the ficoll density gradient on the CFUs content of the irradiated mouse bone marrow, two subpopulations of T-cells were isolated: the administration of the first thymocyte subpopulation decreased the CFUs content during the postirradiation recovery period while thymocytes of the second subpopulation increased the content of CFUs in the bone marrow. When thymocytes of mice exposed to low-level radiation were separated a considerable stimulatory effect was produced by certain thymus cell fractions on the number of CFUs in the bone marrow of exposed recipients; no inhibitory effect was registered.     

Evidence that the Pgp-1 glycoprotein is expressed on thymus-homing progenitor cells of the thymus

The Pgp-1 glycoprotein is found on the bone marrow prothymocyte; however, only a few percent of cells within the normal thymus express significant quantities of Pgp-1 glycoprotein. One hypothesis is that some or all of these Pgp-1+ thymocytes represent thymocyte progenitors or the immediate descendents of the bone marrow-derived prothymocyte. A cell present in the thymus which is able to home back to the thymus and to transiently repopulate it represents one class of thymocyte progenitor. Thymocyte populations enriched in this thymus-homing progenitor are enriched in Pgp-1+ cells. Treatment of these enriched populations with anti-Pgp-1 antibody inhibits activity of the thymus-homing progenitor. These results are consistent with the hypothesis that the thymus-homing progenitor bears Pgp-1 on its surface.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation I. Regeneration and Quantification of Thymocyte Progenitor Cells In the Bone Marrow

Growth kinetics of the donor-type thymus cell population after transplantation of bone marrow into irradiated syngeneic recipient mice is biphasic. During the first rapid phase of regeneration, lasting until day 19 after transplantation, the rate of development of the donor cells is independent of the number of bone marrow cells inoculated. the second slow phase is observed only when low numbers of bone marrow cells (2.5 × 10⁴) are transplanted. the decrease in the rate of development is attributed to an efflux of donor cells from the thymus because, at the same time, the first immunologically competent cells are found in spleen. After bone marrow transplantation the regeneration of thymocyte progenitor cells in the marrow is delayed when compared to regeneration of CFUs. Therefore, regenerating marrow has a greatly reduced capacity to restore the thymus cell population. One week after transplantation of 3 × 10⁶ cells, 1% of normal capacity of bone marrow is found. It is concluded that the regenerating thymus cells population after bone marrow transplantation is composed of the direct progeny of precursor cells in the inoculum.     

Characterization of thymic progenitors in adult mouse bone marrow

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1(-)) (Thy1.1(-) progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1(-) progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1(-) population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.     

ChT1, an Ig superfamily molecule required for T cell differentiation

The thymus is colonized by circulating progenitor cells that differentiate into mature T cells under the influence of the thymic microenvironment. We report here the cloning and function of the avian thymocyte Ag ChT1, a member of the Ig superfamily with one V-like and one C2-like domain. ChT1-positive embryonic bone marrow cells coexpressing c-kit give rise to mature T cells upon intrathymic cell transfer. ChT1-specific Ab inhibits T cell differentiation in embryonic thymic organ cultures and in thymocyte precursor cocultures on stromal cells. Thus, we provide clear evidence that ChT1 is a novel Ag on early T cell progenitors that plays an important role in the early stages of T cell development.     

Differentiation patterns of CD4/CD8 thymocyte subsets in cocultures of fetal thymus and lymphohemopoietic cells from c-fos transgenic and normal mice.

This study examined the involvement of c-fos protooncogene in thymocyte development from lymphohemopoietic T cell progenitors, within the thymic microenvironment. We first analyzed the thymocytes developing in vitro in the fetal thymus from the c-fos transgenic mice and found a high proportion of CD4+ single positive (SP) cells. We then seeded either fetal liver or bone marrow (BM) cells from normal donors onto lymphocyte-depleted fetal thymus explants of c-fos transgenic mice. The results showed an increased proportion of mature CD4+ SP and decreased CD4+CD8+ double positive (DP) cells. A similar pattern of CD4/CD8 thymocyte subsets was observed when either thymus or BM cells from c-fos transgenic mice developed within a normal thymic stroma. The kinetics of thymocyte development in organ culture (from Days 3 to 11) suggested that the SP cells obtained under these conditions may have bypassed the CD4+CD8+ DP phase. It appears that the altered pattern of thymocyte development manifested in adult c-fos transgenic mice can be induced by the early embryonic thymic stroma, and may also involve cells in the lymphohemopoietic tissues.     

Hematopoietic thymocyte precursors. III. A population of thymocytes with the capacity to return ("home") to the thymus.

A subpopulation of thymocytes with the capacity to return (“home”) to and repopulate the thymus of an irradiated mouse has been identified. These cells differ from the majority of cortical thymocytes in that they are of a lower buoyant density and have higher cortisone and X-ray resistance. They also bear an antigen common to mouse brain but not found on the majority of cortical thymocytes. This subpopulation is derived from a hematopoietic thymocyte progenitor and provides an intrathymic reserve of thymocyte precursors. It appears to be a non self-sustaining “transit” population in the pathway of T-cell development.     

The influence of dietary protein deficiency on haemopoietic cells in the mouse.

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1-4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs--although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18%) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstructive role during the refeeding process.     

Terminal deoxynucleotidyl transferase (TdT) enzyme in thymus and bone marrow: I. Age-associated decline of TdT in humans and mice

This study was aimed at characterizing terminal deoxynucleotidyl transferase (TdT) levels in populations of normal human and murine lymphocytes and toward correlating TdT enzyme levels with the biological process of aging. A newly developed method that utilizes a small number of cells was employed to determine TdT levels in bone marrow and thymus cells following cell fractionation at unit gravity sedimentation. By these methods, cell fractions with high TdT activity were found to comprise only 5–10% of the parent cell pools. In the human bone marrow, we show here that TdT-positive cell fractions are largely depleted of HTLA, E-rosette forming, and mitogen-responsive cells, whereas TdT-positive human thymocyte fractions contain a high percentage of HTLA and E-rosette-positive cells. Our observations in the murine model confirm the earlier observations that TdT activity decreases with age. We further show here that the age-associated decline of TdT in the bone marrow preceded that in the thymus. As is true for the mouse, TdT activity in human bone marrow and thymus was also found to decrease with advancing age. The decline in TdT was not associated with a change in cell distribution profiles after unit gravity sedimentation of bone marrow or thymus cells. From these data, the age-associated loss of TdT cannot be attributed to a loss of a particular subpopulation of cells.     

Endotoxin-induced size change in bone marrow progenitors of granulocytes and macrophages

Injection of 5 μg endotoxin to adult C₅₇BL mice caused a marked increase in the sedimentation velocity of granulocytic and macrophage progenitor (colony-forming) cells in the bone marrow. This change was maximal two days after injection and was not accompanied by corresponding changes in total marrow nucleated cell populations. The endotoxin-induced shift was not dependent on the presence of the thymus but did not occur in mice challenged after preinjection with endotoxin. No changes in buoyant density, cell cycle status, pattern of differentiation and responsiveness of granulocytic and macrophage progenitor cells were observed after the injection of endotoxin. The increased sedimentation velocity of progenitor cells appears to indicate an increase in cell volume but the mechanisms involved have not been identified.     

Modification of the regulatory effects of thymocytes on hematopoiesis in irradiated mice as affected by heterologous immunoglobulin G and low-dose ionizing radiation

The administration of heterologous immunoglobulin G (IgG) and/or exposure of mouse thymocyte donors to 1 and 2 Gy radiation were shown to change the regulatory effects of thymus lymphocytes on the recovery of haemopoiesis in syngeneic recipients irradiated with a median lethal dose of 6 Gy. Thymocytes of exposed (2 Gy) donors produced a stimulatory effect on the restoration of the myelokaryocytes number and increased the number of endogenous splenic colonies and bone marrow CFUs in animals exposed to a median lethal dose, whereas the administration of IgG to thymocyte donors given 2 Gy eliminated the stimulatory effect of thymocytes on the number of myelokaryocytes, and the amount of CFUs in irradiated recipients decreased.     

Early ontogeny of thymocytes in pigs: sequential colonization of the thymus by T cell progenitors

Successive colonization of the thymus by waves of thymocyte progenitors has been described in chicken-quail chimeras and suggested from studies in mice. In swine, we show that the first CD3epsilon-bearing thymocytes appear on day 40 of gestation (DG40). These early thymocytes were CD3epsilonhigh and belonged to the gammadelta T cell lineage. Mature CD3epsilonhigh alphabeta thymocytes were observed 15 days later (DG55), and their occurrence was preceded by the appearance of CD3epsilonlow thymocytes (DG45). Thereafter, we observed transient changes in thymocyte subset composition (DG56-DG74), which can be explained by a gap in pro-T cell delivery to the thymus. This delivery gap corresponds with the expression of the pan-leukocyte CD45 and pan-myelomonocytic SWC3a markers in fetal liver and bone marrow and is probably caused by shifting of primary lymphopoiesis between these organs. Therefore, we conclude that the embryonic thymus is colonized by at least two successive waves of hemopoietic progenitors during embryogenesis and that the influx of thymocyte progenitors is discontinuous. Surface immunophenotyping and cell cycle analysis of thymocyte subsets allowed us to compare thymocyte differentiation in pigs with that described for rodents and humans and to propose a model for T cell lymphopoiesis in swine. We also observed that the porcine IL-2Ralpha (CD25), a typical differentiation marker of pre-T cells in mice and humans, was not expressed on thymocyte precursors in pigs and could only be found on mature thymocytes. Finally, we observed a subset of TCRgammadelta+ thymocytes that were cycling late during their development in the thymus.     

Direct BMP2/4 signaling through BMP receptor IA regulates fetal thymocyte progenitor homeostasis and differentiation to CD4+CD8+ double-positive cell

BMP2/4 signaling is required for embryogenesis and involved in thymus morphogenesis and T-lineage differentiation. In vitro experiments have shown that treatment of thymus explants with exogenous BMP4 negatively regulated differentiation of early thymocyte progenitors and the transition from CD4−CD8− (DN) to CD4+CD8+ (DP). Here we show that in vivo BMP2/4 signaling is required for fetal thymocyte progenitor homeostasis and expansion, but negatively regulates differentiation from DN to DP cell. Unexpectedly, conditional deletion of BMPRIA from fetal thymocytes (using the Cre-loxP system and directing excision to hematopoietic lineage cells with the Vav promoter) demonstrated that physiological levels of BMP2/4 signaling directly to thymocytes through BMPRIA are required for normal differentiation and expansion of early fetal DN thymocytes. In contrast, the arrest in early thymocyte progenitor differentiation caused by exogenous BMP4 treatment of thymus explants is induced in part by direct signaling to thymocytes through BMPRIA, and in part by indirect signaling through non-hematopoietic cells. Analysis of the transition from fetal DN to DP cell, both by ex vivo analysis of conditional BMPRIA-deficient thymocytes and by treatment of thymus explants with the BMP4-inhibitor Noggin demonstrated that BMP2/4 signaling is a negative regulator at this stage. We showed that at this stage of fetal T-cell development BMP2/4 signals directly to thymocytes through BMPRIA.     

THE INFLUENCE OF DIETARY PROTEIN DEFICIENCY ON HAEMOPOIETIC CELLS IN THE MOUSE

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1 -4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs–although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18 %) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstitutive role during the refeeding process.     

The blood contains multiple distinct progenitor populations with clonogenic B and T lineage potential

The thymus is seeded by bone marrow-derived progenitors that circulate in the blood. Multiple cell types can be found in the thymus early after i.v. administration or in steady state, but most fail to satisfy the known characteristics of true T progenitors. Cells that do conform to classical definitions retain multilineage potential, but surprisingly, cannot make B cells. Because acquisition of the T lineage fate among noncommitted progenitors is a lengthy process, the absence of B cell potential in early thymocytes suggests that B and T lineages diverge prethymically. To test this suggestion, we screened numerous presumptive progenitor populations for T cell growth and differentiation potential, as well as for clonogenic T or B cell development. We find that blood and marrow each contain multiple distinct subsets that display growth and differentiation potential consistent with being canonical T progenitors. Assessment of clonogenic potential further shows that although all blood and marrow populations have high T cell cloning potential, no T/non-B cells are apparent. These data suggest that either true thymic reconstitution potential derives from a small T/non-B cell subset of one of these populations, or that most of the cells defined as canonical progenitors within the thymus do not, in fact, reside in the mainstream of T progenitor differentiation.     

Induction of erythropoietin responsiveness in vitro by a distinct population of bone marrow cells.

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid 'burst-forming units' (BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.     

INDUCTION OF ERYTHROPOIETIN RESPONSIVENESS IN VITRO BY A DISTINCT POPULATION OF BONE MARROW CELLS

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid ‘burst-forming units’(BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.