Joan Wright Goodman

Influence of thymus cells on erythropoiesis of parental marrow in irradiated hybrid mice.

Summary Living parental thymus cells injected into heavily irradiated F1 hybrid recipients augmented erythropoiesis resulting from transplanted bone marrow from the same, but not the second, parental strain. The augmentation increased as parental thymus cell dose was increased. Thymus cells alone, regardless of origin, gave rise to no erythropoiesis. When recipient spleen weight, 8 days after marrow transplantation, was used as a criterion of marrow growth, the data for parental cell doses 4 × 107 and above paralleled those from 59Fe-uptake studies in the same mice. Parental thymus cells were effective even when administered 1 to 2 days before or after the marrow transplant.

Further studies on the relationship of the thymus to hemopoiesis.

Thymocytes of parental (P) type did not augment hemopoiesis in irradiated hybrid (F1) recipients of P marrow when (1) implanted i.p. in Millipore diffusion chambers as cell suspensions or as tissue slices; (2) disrupted by freezing and thawing or by sonification; or (3) used as saline extracts. These results suggest strongly that a humoral factor is not responsible for the augmenting effect. The augmentation of early marrow growth in P → F1 chimeras, produced by a standard number of P thymocytes, was dependent on age of thymus donor (within the limits of 1–13 weeks in this study). The effectiveness of thymoeytes from donors of different ages was related inversely to cell yield. The ability of thymocytes to augment marrow growth was found in transplantation situations other than the particular P → F1 poor growth cases previously studied so extensively; these include the allogenic A.SW → B6D2F1 and xenogenic Sprague-Dawley rat → B6D2F1 combinations. Ninety-day mortality data from several P → F1 and allogenic marrow plus thymocyte transplantation experiments showed that thymus of marrow donor type, either P or allogenic to F1 recipients, decreased survival of chimeras by accelerating graft-versus-host (GVH) disease. However, all long-term survivors of this constitution, although few in number, retained marrow grafts, whereas those that had been given either host-type or no thymocetes occasionally reverted to host-type red cells.

Spleen Weight as a Measure of Marrow Cell Growth in Irradiated Mice.

Summary C57BL, C3H, C57BL/6, and DBA/2 parental marrow cells were injected at varying doses into lethally irradiated BC3F1 and B6D2F1 mice. Spleen weight in recipients was compared to spleen colony formation and Fe59 uptake. Results showed that increased spleen weight was proportional to formation of increased numbers of splenic nodules and percentage of Fe59 uptake. Data suggest that spleen weight, spleen colony formation, and Fe59 uptake are equally suitable assays, within appropriate limits, for studies on proliferation of marrow cell transplants in irradiated mice. Spleen weight is a useful index of growth for doses of transplanted marrow cells which promote recovery of hematopoietic injury in lethally irradiated mice.

DONOR-TYPE LYMPHATIC TISSUE CELLS IN LETHALLY IRRADIATED MICE TREATED WITH HOMOLOGOUS FETAL LIVER

Normal lymph node structure was found in lethally irradiated LAF/sub 1/ mice treated with homologous 101C3F/sub 1/ fetal liver cells. Specific cytotoxic antisera prepared against host-type cells failed to react with bone marrow and lymph node cells. The conclusion is drawn that these cells are of donor origin. (auth)

THE USE OF ISOIMMUNE SERA FOR IDENTIFICATION OF HEMOPOIETIC CELLS FROM BLOOD‐INJECTED RADIATION CHIMERAS

Use has been made of the antigenic differences among mice with respect to both erythrocytes and nucleated cells to identify hemopoietic cells of radiation chimeras. By employing isoimmune sera in agglutination and cytotoxic techniques, it has been shown that long-term leukemoid blood chimeras contain lymphocytes, erythrocytes, and nucleated bone marrow cells of the blood-donor type; blood from normal mice can repopulate lymphoid tissues of radiationbone marrow chimeras, and the blood-derived cells might take part in graft-versus-host immune reactions; and normal peripheral leukocytes from F/sub 1/ hybrid donors can reestablish lymphopoiesis, granulopoiesis, and erythropoiesis when injected into lethally irradiated parent strain mice. Cytotoxic typing of the nucleated hemopoietic cells shows that they are of donor origin. (auth)

ABILITY OF THYMIC LYMPHOCYTES TO ALTER CFU KINETICS IN RADIATION CHIMERAS

It is known that the poor colony‐forming ability of B6 bone marrow transplanted into B6D2F1 hybrids can be improved if B6 lymphocytes are given in addition. It was recently reported that the augmenting lymphocytes decrease the doubling time of differentiating hemopoietic cells. to determine whether thymus cells alter the self‐renewal of CFUs in this parent F1 combination, retransplan‐tation and 3H‐thymidine ‘suicide’ were employed as methods to determine the cell‐division rate. We have observed that in the presence of thymocytes, parental bone marrow cells are seeded more efficiently in the spleen, and the lag phase of the CFUs growth curve is shortened. However, thymic lymphocytes do not increase the slope of the exponential growth phase of CFUs.

Bone marrow cell separation studies

Abstract Differential gradient centrifugation of bone marrow cell suspensions in bovine serum albumin produced layers of mixed cell types. Each layer was capable isologously of promoting survival of heavily irradiated (C57BL × 101)F 1 mice; none was more effective than fresh whole bone marrow. Survival curves at various nucleated cell doses were established for the several lavers and for whole marrow. The effective cell type or types in this system were remarkably resistant to various kinds of trauma, including the prolonged procedures of centrifugation, layer cutting, and resuspension involved in the gradient separation, and also exposure for as long as 4 minutes to distilled water. Donor mice were pretreated by several methods, including production of artificial polycythemia and injection of cobalt, cortisone, and corticotropin in attempts to alter marrow populations. The marrow, when tested in lethally irradiated mice, was at least as effective as normal marrow. Qualitatively, the separated layers obtained on differential gradient centrifugation of such pretreated marrows appeared no different from “normal” layers. Various substances used in these and other routine bone marrow procedures in this laboratory were nontoxic to bone marrow cells under the conditions of the bioassay used.

Effects of isogenic bone marrow cell dose on erythrokinetics of lethally x-irradiated mice.

The effect of isogenic bone marrow cell dose on several parameters of hemopoietic regeneration in lethally X‐irradiated mice was studied. The appearance of 59Fe in red blood cells (RBC) and spleen, the hematocrits, and the spleen weights were measured at various times after irradiation and the injection of 105, 106, 107 or 108 marrow cells. Results show that with increasing cell dose, regeneration of erythropoiesis was detectable earlier, proceeded at a faster rate, reached a peak more quickly, and subsided to normal levels sooner. The initial spleen‐weight growth rate was independent of cell dose, although the onset of growth was earlier the higher the dose. The maximum rate of RBC production was attained earlier the larger the cell dose and was a decreasing function of the hematocrit at that time, which in itself was an increasing function of cell dose. These data support the concept of a proportional model for control of erythropoiesis as described by Hodgson (1970).

Effect of thymus transplantation on radiation chimeras.

Recent evidence points to the thymus of the neonatal mouse as the embryologic source of stem cells for the immune mechanism of the adult animal, even though the fetal thymus itself lacks immunological competence (Auerbach, 1961; Miller, 1961, 1962; Martinez, 1962). There is no reason to think that the large thymus of the young adult animal, or even the atrophic thymus of the old animal, does not continue to be a significant factor in the function of the immune mechanism (Congdon and Duda, 1961; Swartzendruber et al., 1961; Congdon and Doherty, 1963; Miller, 1962). Evidence that the adult thymus might disburse its cells to function immunologically in other sites, such as spleen and liver, has been reported by Fichtelius (1953). Four different kinds of protection and recovery experiments are pertinent to the study of thymic function in the young adult mouse. The first of these concerns chemically protected animals. In lethally irradiated mice protected with AET, thymus regeneration precedes by several days regeneration of the other lymphatic tissues, thus allowing one to speculate that the thymus in the adult animal might be the source of the regenerated tissue. These findings were based on histologic evidence of recovery rather than tissue weight changes (Congdon and Doherty, 1963). In another type of experiment, injection of isologous thymus cells into normal mice did not produce detectable histologic changes in the host thymus or the lymphatic tissues. However, in irradiated mice marked accumulation of the thymocyte-like cells appeared in the spleen red pulp after intravenous injection of many millions of thymus cells. When an antigenic stimulus was provided, many antibody-forming cells developed in the red pulp, perhaps by transformation from the thymus cells (Congdon and Duda, 1961). In a third experiment, direct identification of thymus cells in rat bone marrow chimeras was made. It was noted that the initial regeneration of thymocytes was of mouse host type, according to the identification system used (Gengozian et al., 1957). Later, only thymocytes of rat type were identified. It is conceivable these could then give rise to a rat im-

Origin of cells repopulating the spleens of irradiated mice given marrow cells and thymocytes

SUMMARY Hemopoietic tissues are poorly repopulated in lethally irradiated mice by bone marrow transplanted from certain homologous or heterologous animals. Administration of marrow donor‐type thymocytes has been shown to augment repopulation. We have confirmed augmentation of repopulation, using mitotic indices of spleens 8 days after injection of bone marrow cells or thymocytes or both, and we have shown that most of the repopulating cells are derived from the donor marrow. Using female rats as bone marrow donors and male rats as thymus donors, and vice versa, we found that 93.1% of the dividing cells in repopulated mouse spleen were of rat bone marrow origin. About 1.0% of the dividing cells were of mouse origin, and 5.9% were derived from rat thymus.