Repopulation of the mouse thymus after sublethal fission neutron irradiation. II. Sequential changes in the thymic microenvironment

The stromal cells of the thymus of sham-irradiated and sublethal fission neutron-irradiated CBA/H mice were analyzed with immunohistology, using monoclonal antibodies directed to I-A and H-2K antigens as well as specific determinants for cortical and medullary stromal elements. In the control thymuses, I-A expression in the thymus shows a reticular staining pattern in the cortex and a confluent staining pattern in the medulla. In contrast, H-2K expression is mainly confluently located in the medulla. Whole body irradiation with 2.5 Gy fission neutrons reduces within 24 hr the cortex to a rim of vacuolized "nurse cell-like" epithelial cells, largely depleted of lymphoid cells. The localization of I-A antigens changes in the cortex and I-A determinants are no longer associated with or localized on epithelial reticular cells. Medullary stromal cells, however, are more or less unaffected. A high rate of phagocytosis is observed during the first 3 days after irradiation. About 5 days after irradiation, the thymus becomes highly vascularized and lymphoid cells repopulate the cortex. The repopulation of the thymic cortex coincides with the appearance of a bright H-2K expression in the cortex which is associated with both stromal cells as well as lymphoid blasts. During the regeneration of the thymus, the thymic stromal architecture is restored before the expression of cell surface-associated reticular MHC staining patterns. The observed sequential changes in the thymic microenvironment are related to the lymphoid repopulation of the thymus.     

Nonrandom distribution of mouse spermatogonial stem cells surviving fission neutron irradiation

Colony formation by surviving spermatogonial stem cells was investigated by mapping pieces of whole mounted tubuli at intervals of 6 and 10 days after doses of 0.75 and 1.50 Gy of fission neutron irradiation. Colony sizes, expressed in numbers of spermatogonia per colony, varied greatly. However, the mean colony size found in different animals was relatively constant. The mitotic indices in large and small colonies and in colonies in different epithelial stages did not differ significantly. This finding suggests that size differences in these spermatogenic colonies are not caused by differences in growth rate. Apparently, surviving stem cells start to form colonies at variable times after irradiation. The number of colonies per unit area varied with the epithelial stages. Many more colonies were found in areas that during irradiation were in stages IX-III (IX-IIIirr) than in those that were in stages IV-VII (IV-VIIirr). After a dose of 1.50 Gy, 90% of all colonies were found in areas IX-IIIirr. It is concluded that the previously found difference in repopulation after irradiation between areas VIII-IIIirr and III-VIIIirr can be explained not by differences in colony sizes and/or growth rates of the colonies in these areas but by a difference in the number of surviving stem cells in both areas. In area XII-IIIirr three times more colonies were found after a dose of 0.75 Gy than after a dose of 1.50 Gy. In area IV-VIIirr the numbers of colonies differed by a factor of six after both doses. This finding indicates that spermatogonial stem cells are more sensitive to irradiation in epithelial stages IV-VII than in stages XII-III. In control material, spermatogonia with a nuclear area of 70-110 micron2 are rare. However, especially 6 days after irradiation, single cells of these dimensions are rather common. These cells were found to lie at random over the tubular basement membrane with no preference for areas with colonies. It is concluded that the great majority of these cells were not or do not derive from surviving stem cells. These enlarged cells most likely represent lethally injured cells that will die or become giant cells (nuclear area greater than 110 micron2).     

Studies on thymocyte subpopulations in guinea pigs. X. Rosette-forming ability of thymocyte subpopulations before and after incubation in vitro

Thymocytes spontaneously proliferating in vitro were labelled with 3H-thymidine, and the distribution of label among rosette-forming cells (RFC+) and nonrosetting cells (RFC-), as well as in populations differing in buoyant density, was measured by liquid scintillation counting and autoradiography before and after incubation for 24 h. Initially most labelled cells (88%) belonged to the low-density (1a) subpopulation, the majority being RFC+. After incubation for 24 h, low-density nonrosetting thymocytes (1a, RFC-) contained the highest amount of label. A decreased rosette formation occurred not only in labelled cells but also in the population as a whole, and in separately incubated high-density cells. The decreased rosette formation was mainly caused by a change in rosette-forming ability of viable high-density cells, however in part also by decreased viability. A shift from low to high density occurred among labelled cells during incubation and was shown to occur in both RFC+ and RFC-. The decreased rosette formation of labelled cells during in vitro culture contrasts with the increase earlier observed in vivo and may therefore represent affinity alterations or a down-regulation of the rosette receptor in vitro. We conclude that the observed changes in density, but not in rosette-forming ability, may reflect normal differentiation.     

The differentiation of T lymphocytes. I. Proliferation kinetics and interrelationships of subpopulations of mouse thymus cells

Differential quantitative cytotoxic assays were used to distinguish the major high θ population of mouse thymus from the minor low θ subpopulation. The low θ cells were isolated in good yield by killing all high θ cells with controlled anti-θ and complement treatment, followed by a damaged cell removal step. A third population of labile cells, subject to rapid death in culture, was distinguished as a variable component within the high θ category. The corticosteroid sensitivity and anatomical location of these subpopulations was briefly considered. Large and medium sized dividing lymphocytes were studied by pulse labeling with tritiated thymidine, followed by radio-autography of separated subpopulations. Both the high θ and low θ categories included large dividing cells. Kinetic studies under conditions of continuous labeling were used to explore precursor-product relationships among the small thymocytes. Both low θ and high θ small lymphocytes showed continuous and close to linear accumulation of labeled cells, with no evidence for a marked lag in labeling. The turnover time of high θ small lymphocytes was three–four times that of low θ elements. The results suggest largely independent pathways are involved in the development of the two antigenically defined subpopulations. They do not support a direct transfer of “immature” high θ, TL positive small thymocytes in mature, active, low θ,TL negative cells. Some alternative models of T cell development are discussed.     

Repopulation of the seminiferous epithelium of the rhesus monkey after X irradiation

Repopulation of the seminiferous epithelium became evident from Day 75 postirradiation onward after doses of 0.5, 1.0, and 2.0 Gy of X rays. Cell counts in cross sections of seminiferous tubules revealed that during this repopulation the numbers of Apale (Ap) spermatogonia, Adark (Ad) spermatogonia, and B spermatogonia increased simultaneously. After 0.5 Gy the number of spermatogonia increased from approximately 10% of the control level at Day 44 to 90% at Day 200. After 1.0 and 2.0 Gy the numbers of spermatogonia increased from less than 5% at Day 44 to 70% at Days 200 and 370. The number of Ad and B spermatogonia, which are considered to be resting and differentiating spermatogonia, respectively, already had increased when the number of proliferating Ap spermatogonia was still very low. This early inactivation and differentiation of a large part of the population of Ap spermatogonia slows down repopulation of the seminiferous epithelium of the primates. By studying repopulating colonies in whole mounts of seminiferous tubules various types of colonies were found. In colonies consisting of only A spermatogonia, 40% of the A spermatogonia were found to be of the Ad type, which indicates that even before the colony had differentiated, 40% of the A spermatogonia were inactivated into Ad. Differentiating colonies were also found in which one or two generations of germ cells were missing. In some of those colonies it was found that the Ap spermatogonia did not form any B spermatogonia during one or two cycles of the seminiferous epithelium, while in other colonies all Ap spermatogonia present had differentiated into B spermatogonia. This indicates that the differentiation of Ap into B spermatogonia is a stochastic process. When after irradiation the density of the spermatogonia in the epithelium was very low, it could be seen that the populations of Ap and Ad spermatogonia are composed of clones of single, paired, and aligned spermatogonia, which are very similar to the clones of undifferentiated spermatogonia in non-primates.     

Repopulation of spleens of irradiated mice after injection of spleen cell subpopulations

Abstract. Haemopoietic stem cells present in the spleen of adult mice were analysed by grafting X-irradiated animals with polystyrene-nonadherent (NABS) and polystyrene-adherent (ABS) B-enriched splenocytes from syngeneic donors. The progeny of the haemopoietic stem cells present in NABS and ABS subsets were studied with respect to size, surface markers, and response to mitogens and antigens. Ninety-six per cent of the precursors of the myeloid cell lineage (CFU-S) were present in the NABS fraction (50-fold enrichment). The presence in NABS of progenitors of functional T and B lymphocytes was also demonstrated. Twelve days after grafting with NABS, more than 80% of the recipient splenocytes were large and nonadherent granulocyte-like cells. These cells had surface similarities with NABS from normal mice, since both populations reacted with peanut agglutinin and with a rabbit anti-NABS (RAN) serum.     

Characteristics of mouse hematopoietic stem cells in the phase of enhanced radioresistance after sublethal irradiation

Haemopoietic stem cells of mice which displayed an increased radioresistance 12 days following sublethal irradiation, exhibited higher proliferative, granulocyte-repopulating and migration activities as compared with those of intact animals.     

Studies on the termination of immunological tolerance in the mouse thymus cell population after irradiation

Immunological tolerance in the mouse thymus cell population induced by the intravenous injection of deaggregated bovine gamma globulin was terminated by whole body irradiation. After irradiation, the weight of the thymus recovered biphasically, and the termination of tolerance occurred as early as in the first phase. Both Thy-1 antigen expression and helper activity of the thymus cell population in irradiated mice recovered in parallel with the recovery of the thymus weight. Sensitivity of the regenerating thymus cell to the tolerogen was not different from that of the normal thymus cell. The first phase of thymus regeneration may be caused by the proliferation and differentiation of relatively radioresistant and tolerogen insensitive precursors residing in the thymus. Tolerogen and/or immunogen reactive thymus cells may originate from the precursor.     

Probability of self-renewing divisions of spermatogonial stem cells in colonies, formed after fission neutron irradiation

Abstract.
Repopulating spermatogenic colonies, found in the seminiferous epithelium after irradiation with fast-fission neutrons, were studied to determine the cha nce that a stem cellA_single (A_s spermatogonium would complete a self-renewing division (P). Mathematical formulas originally derived for such studies in haemopoietic colonies were employed, and a method specifically aimed at spermatogenic colonies was developed. The results showed that during the first division after irradiation, P is close to 1 -0. P decreases in later generations, but remains 0-7 or higher up to the 4th or 5th divisions. The mean value for P was over 0-8, which is higher than the value of 0-6-0-7 found for stem cells in haemopoietic colonies.     

The cell cycle of spermatogonial colony forming stem cells in the CBA mouse after neutron irradiation

In the CBA mouse testis about 10% of the stem cell population is highly resistant to neutron irradiation (D0, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea it appeared that in this cycle the S-phase is less radiosensitive (D0, 0.43 Gy) than the other phases of the cell cycle (D0, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. The data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most.     

Unilateral T cell maturation arrest in the thymus of CBA/H mice as a long-term effect after neutron irradiation

Thymuses of CBA/H mice were investigated up to 570 days after whole-body irradiation with 2.5 Gy fast fission neutrons or 6.0 Gy X rays. A number of these thymuses, observed 220-270 days after neutron irradiation, have two equal sized lobes, one of which has an abnormal T cell distribution. The present paper reports on the distribution of lymphoid and stromal cell types in these thymuses. For this purpose, we employed immunohistology using the indirect immunoperoxidase method. We incubated frozen sections of these aberrant thymuses with monoclonal antibodies directed to cell surface differentiation antigens on lymphoid cells, such as Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14; monoclonal antibodies directed to major histocompatibility complex (MHC) antigens, such as I-A and H-2K; and monoclonal antibodies directed to determinants in various thymic stromal cell types. The results of this study show a T cell differentiation arrest in only one of the two thymic lobes. T cells in the aberrant lobe express Thy-1, T-200, and MEL-14 antigens but are MT-4- and Lyt-1-. In some lobes, a weak Lyt-2 expression was observed. The observed T cell maturation arrest is mainly restricted to the cortex since in the medulla, in addition to cells with an aberrant cortical phenotype, normal T cell phenotypes are observed. This indicates that cortex and medulla have independent generation kinetics in T cell maturation. The stromal cell composition in these abnormal lobes is not different from that in the normal lobe, but the size of the medulla tends to be smaller. Furthermore, the I-A expression on the cortical epithelial cells does not reveal the characteristic reticular staining pattern that is observed in the normal lobe, since the I-A determinants are not strictly confined to the epithelial cells. In addition, cortical lymphoid and stromal cells in these lobes are slightly H-2K+. These alterations in MHC expression in the cortex are discussed in relation to the observed T cell maturation arrest.     

Protection of mice against fission neutron irradiation by WR-2721 or WR-151327

Two phosphorothioate compounds, WR-2721 and WR-151327, were examined for their radioprotective efficacies against the effects of fission neutron irradiation in male and female mice. Within sex groups no significant difference in lethality at 30 or 100 days postirradiation was found between WR-2721 or WR-151327 pretreatment. The dose modification factors (DMFs) for male mice treated with either compound were 1.29 (LD50/30) and 1.24 (LD50/100), and those for drug-treated female mice were 1.21 (LD50/30) and 1.19 (LD50/100). Both WR-2721 and WR-151327 were found to be equally radioprotective when compared using DMFs as the end point. WR-151327 (500 mg/kg, ip) was found to be significantly more toxic to both male and female B6D2F1 mice than equimolar amounts of WR-2721. Small but significant sex differences in radioprotection were found: the DMFs for female mice pretreated with either compound were lower than those for similarly treated male mice; the incidence of mortality 31-100 days postexposure in male mice pretreated with WR-151327 was greater than for female mice. In addition, sex differences were noted in drug toxicity. Toxic death in female mice given WR-151327 (500 mg/kg, ip) is 2.6 times more probable than in males.