Abnormal stem cells in autoimmune-prone mice are responsible for premature thymic involution

Autoimmune-prone mice show premature thymic involution, including morphological and functional abnormalities. To determine why the thymic abnormalities develop in autoimmune-prone mice, transplantation of the thymus and/or bone marrow was performed. When thymuses of newborn MRL/1 (H-2k) mice were grafted into C3H/HeN nu/nu(H-2k) mice, the engrafted thymuses did not show the abnormalities which characterize the thymus in the autoimmune-prone MRL/1 mice. By contrast, when thymuses of newborn C3H/HeN or MRL/n mice were grafted into MRL/1 mice, the engrafted thymuses developed after an interval of 3 months the same morphological abnormalities as were seen in MRL/1 mice. Thus, we can conclude that premature involution of the thymus in autoimmune-prone mice may not be a genetically determined abnormality intrinsic to the thymus, but rather an abnormality secondary to other events occurring in these mice. When bone marrow of young C3H/HeN nu/nu mice was transplanted into irradiated (850 rad) MRL/1 mice, neither thymic abnormalities nor autoimmune diseases developed. Therefore, it seem likely that abnormal stem cells in autoimmune-prone mice induce thymic abnormalities, and these, in turn, are associated with the development of autoimmune diseases.     

Thymic nurse cells and thymic repopulation after whole body sublethal irradiation in mice

Thymic Nurse Cells (TNCs) are lymphoepithelial complexes which are thought to play a role in the early stages of the intrathymic differentiation pathway. Therefore, their repopulation kinetics were analyzed in mice after sublethal whole-body irradiation. Changes of the number of TNCs per thymus were parallel with the evolution of the whole thymocyte population. Particularly, a first wave of TNCs restoration was followed by a secondary depletion and a final recovery. This suggests that TNCs restoration is related to the proliferating progeny of intrathymic radioresistant thymocytes. When normal bone marrow cells were grafted intravenously after irradiation, no secondary depletion was found. This pattern of restoration was obviously related to thymic repopulation by cells which were derived from the inoculated bone marrow. Homing studies with FITC labelled bone marrow cells showed that inoculated bone marrow cells did not penetrate TNCs early after irradiation. Later on, when immigrant cells started to proliferate, they were found preferentially within TNCs before spreading in the whole thymus. The results indicate that interactions between immature thymocytes and epithelial cells within TNCs are critical for the first steps of intrathymic lymphopoiesis.     

Influence of age on the proliferation and peripheralization of thymic T cells

Bone marrow cells obtained from B10.Thy-1.1 mice (H-2b, Thy-1.1) were injected directly into the thymus of C57BL/6 mice (H-2b,Thy 1.2) of various ages. Thymocyte precursors in the injected donor-bone marrow cells could proliferate in the thymic microenvironment in the following manner: first, preferentially proliferating into the subcapsular cortex; and second, spreading to the whole layer of the cortex, a portion of them gradually moving into the medulla. The proliferation of donor-type thymocytes was most pronounced when intrathymic injection of bone marrow cells (ITB) was performed in newborn mice and especially prominent in week-old mice; it took approximately ten weeks for donor-type thymocytes to finish the whole course of proliferation, differentiation, and emigration to the periphery. When ITB was performed in mice 4 weeks of age and older, the proliferation of donor-type thymocytes was retarded at onset, less pronounced in magnitude, and disappeared earlier. Emigration of donor-type T cells from the thymus to the peripheral lymphoid tissues occurred most rapidly when ITB was performed in newborn mice, and these T cells continued to reside thereafter in the peripheral lymphoid tissues. However, when ITB was performed in mice 4 weeks of age and older, the number of emigrated T cells in the spleen decreased (about a tenth of that in newborn mice) and, moreover, these T cells resided only transiently in the spleen. It was suggested that T cells emigrating from the thymus of mice from newborn to 2 weeks of age are long-lived, whereas those from the thymus in mice 4 weeks of age and older are short-lived. However, when 4-week-old young adult mice were treated by irradiation or hydrocortisone, the thymic capacity was enhanced in terms of proliferation and peripheralization of thymocytes, and emigrated T cells became long-lived.     

Combined grafting of bone marrow and thymus, and sequential multiple thymus graftings in various strains of mice. The effect on immune functions and life span

The combined grafting of young bone marrow and newborn thymus performed in old mice was effective in restoring the impaired immune functions, but the same treatment performed in middle-aged adult mice had no effect on the life span of C3H/MTV female mice. Sequential multiple newborn thymus graftings starting at young adult age were effective in enhancing immunological functions, delaying the onset of tumor and extending the survival rate to certain degree in the first half of the experimental course in both C3H/MTV as well as C57BL/6 mice, but these effects were not observed in the latter half of the experimental course. It was suggested that multiple newborn thymuses sequentially implanted into the peritoneal cavity underwent atrophy and these atrophic thymuses had a suppressive effect on the host immune system. In autoimmune prone B/WF1 mice, however, the combined grafting of young bone marrow and newborn thymus resulted in suppression of antibody formation to SRBC, and single grafting of either young bone marrow or newborn thymus resulted in a trend of increase in the antibody formation to SRBC. The sequential multiple newborn thymus graftings in B/WF1 mice brought about aggravation of kidney diseases and shortening of the mean life span. On the contrary, administration of thymosin in B/WF1 mice resulted in amelioration of kidney disease and elongation of the mean life span.     

Generation of anti-NZB red blood cell antibody-forming plasma cells from bone marrow cultures of syngeneic and allogeneic mice: functional modulation of helper T-cell subsets in autosensitization.

An in vitro anti-NZB red blood cell (RBC) autoantibody-forming system was developed by culturing young (antiglobulin negative) or old (antiglobulin positive) NZB mouse bone marrow cells in the presence of young or old NZB thymocyte homogenates (or RNA thymus extracts) and young NZB-RBC. Using young NZB bone marrow cells, the greatest number of autoantibody forming cells were seen following stimulation with a mixture of young and old thymocyte homogenates (TH). A higher response was seen with old NZB bone marrow cells stimulated by old NZB thymocyte homogenates and RBC. Phenol-extracted thymic RNA retained activity when added to bone marrow culture containing NZB-RBC. Thymus RNA from 9 to 10 days old NZB mice had no activity although their bone marrow cells responded well to stimulation by young and old thymic RNA together with RBC. By analysing results obtained using T-cells enriched for helper and suppressor activities, we have concluded that abrogation of self-tolerance in mice is due to the appearance of functionally modulated helper T-cell subsets and to imbalance between helper and suppressor T cells.     

Age-related changes of thymus--morphological and functional aspects.

The thymus involutes progressively throughout life, beginning at around the sexual maturation. In long-lived BC3F1 hybrid mice, the thymic capacity to induce T cell differentiation begins to decline earlier than the onset of thymic involution, although the magnitude of the decline is different by the subpopulation of T cells. Morphologically, the most active secretory structure seems to be limited exclusively to the neonatal thymus and certain structural changes, reflective of a decline in secretory function, can be detected early in life and they become more pronounced with age. Heterogeneity of thymic epithelial cells is suggested by the facts that age-related and radiation induced decline of immune activities are different in degree by subpopulation of T cells, and the concept is also supported from a morphological viewpoint. It is thus apparent that age-related thymic involution results in decrease of recruitment of fresh capable T cells and increase of old exhausted T cells, eventually bringing about T cell insufficiency in the aged individuals. Such an impaired immune function in the aged mice can be effectively restored by the combined grafting of young bone marrow and newborn thymus, and the thymus is apparently the most limiting factor in the aged. The biological signficance of age-related thymic involution is also discussed.     

Patterns of dual lymphocyte development in co-cultures of foetal thymus and lymphohaemopoietic cells from young and old mice.

Patterns of lymphocyte development in the thymus were analysed, focusing on newly emigrating bone marrow (BM) and resident thymic cells. We co-cultured foetal (Day 15 of gestation) thymic explants (FT, C57BL/Ka, Thy-1.1), with BM cells from young (2-3 months) or old (24 months) syngeneic, Thy-1 congenic (C57BL/6J, Thy-1.2) mice. When the FT was severely depleted [treated with either 2-deoxyguanosine (dGua) or exposed to an irradiation dose of 20 Gy] BM-type T lymphocytes were dominant, regardless of BM donor age. When the FT was only partially depleted of its proper lymphoid cells (by exposure to 10 Gy), the lymphocytes which developed were from both BM and FT origins, yet the level of donor-type thymocytes from the young mice was higher than that of the old. Under these conditions the proportion of FT-derived double-positive CD4+ CD8+ (DP) cells was higher, and that of single-positive CD4- CD8+ cells was lower, than in the BM-derived cells, irrespective of the BM donor age. The proportions of old BM-derived DP cells were lower than in the young. Co-cultures of thymus cells from young and old mice with partially depleted FT explants resulted in similar proportions of CD4/CD8 subsets from both donor and FT origins, with the exception that in the presence of old-thymus cells there was an increase in the level of FT-type CD4- CD8+ cells. Patterns of T-cell differentiation in the thymus thus seem to be determined by newly emigrating cells and the resident thymocytes.     

Hemopoietic and lymphoid organs in AKR/JY mice with thymic lymphoma

Cytological and histological studies of the thymus, bone marrow, peripheral blood, spleen, and lymph nodes in 6-9-month-old AKR/JY mice showed that the risk of thymic lymphoma in these animals increased with age. Generalization of the tumor process was observed in mice aged over 8 months and first involved the spleen, then the lymph nodes, bone marrow, and liver. A case of primary lymphoma in the spleen was detected.     

AGE-RELATED CHANGES OF THYMUS – MORPHOLOGICAL and FUNCTIONAL ASPECTS –

The thymus involutes progressively throughout life, beginning at around the sexual maturation. In long-lived BG3F₁ hybrid mice, the thymic capacity to induce T cell differentiation begins to decline earlier than the onset of thymic involution, although the magnitude of the decline is different by the subpopulation of T cells. Morphologically, the most active secretory structure seems to be limited exclusively to the neonatal thymus and certain structural changes, reflective of a decline in secretory function, can be detected early in life and they become more pronounced with age. Heterogeneity of thymic epithelial cells is suggested by the facts that age-related and radiation induced decline of immune activites are different in degree by subpopulation of T cells, and the concept is also supported from a morphological viewpoint. It is thus apparent that age-related thymic involution results in decrease of recruitment of fresh capable T cells and increase of old exhausted T cells, eventually bringing about T cell insufficiency in the aged individuals. Such an impaired immune function in the aged mice can be effectively restored by the combined grafting of young bone marrow and newborn thymus, and the thymus is apparently the most limiting factor in the aged. The biological significance of age-related thymic involution is also discussed.     

Immune manipulations and brain-reactive antibody formation in aging mice

Previous studies have demonstrated an increase in brain-reactive antibodies (BRA) in sera of various mammals as a function of age. The present study attempts to obtain a better understanding of the mode of formation of BRA by using various immunologic manipulations in young and old C57BL/6 mice. The mean BRA levels were significantly higher in older mice than in the young ones. Mixtures of bone marrow cells and spleen cells from old donors were able to adoptively transfer high BRA levels in young irradiated mice and this was not altered by pretreatment of the cells with anti-Thy-1.2 or anti-Ig serum plus complement. Transfer of young cells to old irradiated hosts did not lower the high BRA levels in the hosts. However, when the old hosts were also thymectomized and grafted with neonatal thymuses, BRA levels were significantly lowered. The studies tend to indicate that thymic involution or loss of thymic function in aging plays an important role in BRA formation in sera of aged mice.     

Thymocyte progenitors in ageing

The effect of ageing on thymocyte progenitors in the bone marrow was studied in an in vitro experimental model that permits T lymphocyte development. The model is based on co-culture of BM cells from young and old mice with lymphoid depleted fetal thymus explants. We applied different strategies of thymic colonization, including competitive colonization by BM cells from different donor age groups and MHC backgrounds. Our data reveal intrinsic changes in the BM that lead to manifestation of immunosenescence in the T lymphocyte compartment.     

Cell‐autonomous role of EphB2 and EphB3 receptors in the thymic epithelial cell organization

The role of EphB2 and EphB3 in the organization of thymic epithelial cells has been studied in EphB‐deficient fetal thymus lobes grafted under the kidney capsule of WT mice. The deficient lobes, as compared with WT ones, showed altered distribution of medullary areas, shortening of medullary epithelial cell processes and presence of K5^?K8^? areas. EphB2 and EphB3 expressed on thymic epithelial cells play an autonomous role in their organization. The relevance of Eph/ephrinB forward and reverse signals for this process was evaluated in grafted fetal thymus lobes from mice expressing a truncated EphB2 receptor capable of activating reverse, but not forward, signaling. These deficient lobes showed important alterations of the thymic epithelial organization as compared with the grafted WT lobes, but a less severe phenotype than the grafted EphB2‐deficient thymus lobes, which confirms the relevance of EphB2 forward signal for the thymic epithelial organization but, also, a role of the reverse signaling in determining the final epithelial phenotype.     

Age-Related Changes in Primary and Secondary Immune Organs of the Mouse

The present work describes the murine immune tissue evolution with age with special emphasis on the bone marrow. To that effect we monitored the weights of the thymus, spleen and axillary lymph nodes over the first year of life in C57BL/6 male and female mice. In addition, we monitored the relative proportions of erythroid, lymphoid and myeloid cells in the bone marrow, and performed in vitro migration assays of bone marrow cells to thymic supernatants, with the aim of determining whether the migration of such cells or the thymic attractive capacity are affected by age. Before puberty, a remarkable decline in the relative weight of the thymus, spleen and lymph nodes was observed; after that stage, however, only the thymus showed an involution. The proportion of myeloid cells in the bone marrow showed an increase with age. Furthermore, the migration of myeloid cells to thymic supernatants increased with age and paralleled the time-course of the myeloid cell increase found in the bone marrow. More interestingly, the proportion of lymphoid cells to total bone marrow cells showed a clear decline with age. The time-course of this decline closely paralleled that of thymus weight, suggesting that the involution of the thymus may be related to changes in the cell composition of the bone marrow.     

Bone marrow-thymus axis in senescence

This presentation offers a brief review of the bone marrow-thymus axis in senescence, a putatitive model for thymocyte differentiation, and recent results of our work on the status of pre-thymic stem cells in aged mice. The data presented here provide further evidence for a thymus endocrine influence on the bone marrow stem cells, specifically lymphocyte precursors. It has been pstulated that the thymic hormones may act on lymphocyte precursors in the bone marrow and that the loss of thymic factors during senescence may be a contributing factor to the decreased cellular immune function. This study used Haar's in vitro model to investigate the bone marrow-thymus axis in aged mice. Erythroid-depleted bone-marrow cells from 3-month- and 24-month-old CBA (Thy 1.2) mice were placed in the upper half of a blind-well chamber with thymus supernatant in the lower half. Experimental cells were treated with thymus supernatant for 1 hr prior to migration. This study confirmed that pre-thymic stem cells in aged bone marrow are deficient in their ability to migrate to the thymus supernatant. It also revealed that treatment of the old bone marrow with thymus supernatant, made from neonatal thymus cultures, could dramatically improve the thymus migrating ability of the aged bone-marrow stem cells.     

Age-Related Changes in Primary and Secondary Immune Organs of the Mouse

The present work describes the murine immune tissue evolution with age with special emphasis on the bone marrow. To that effect we monitored the weights of the thymus, spleen and axillary lymph nodes over the first year of life in C57BL/6 male and female mice. In addition, we monitored the relative proportions of erythroid, lymphoid and myeloid cells in the bone marrow, and performed in vitro migration assays of bone marrow cells to thymic supernatants, with the aim of determining whether the migration of such cells or the thymic attractive capacity are affected by age. Before puberty, a remarkable decline in the relative weight of the thymus, spleen and lymph nodes was observed; after that stage, however, only the thymus showed an involution. The proportion of myeloid cells in the bone marrow showed an increase with age. Furthermore, the migration of myeloid cells to thymic supernatants increased with age and paralleled the time-course of the myeloid cell increase found in the bone marrow. More interestingly, the proportion of lymphoid cells to total bone marrow cells showed a clear decline with age. The time-course of this decline closely paralleled that of thymus weight, suggesting that the involution of the thymus may be related to changes in the cell composition of the bone marrow.     

The role of bone marrow and thymic elements in the initiation and spread of virus production in the AKR thymus

Passive anti-viral immunotherapy greatly suppresses the incidence of spontaneous leukemia in AKR mice, rendering the thymus of successfully treated animals devoid of infectious ecotropic retrovirus. Reconstitution assays have determined that the thymic and splenic homing cells of the AKR bone marrow become ecotropic virus producers subsequent to their seeding of these hematopoietic organs and that in vitro depletion of gp71 expressing bone marrow cells reduces stem cell numbers without affecting prothymocyte content. In the thymus, a population of radioresistant cells, which phenotypically resemble cortical thymocytes, but are unique in their expression of high levels of H-2Kk antigen, have been found to produce high levels of both ecotropic and MCF virus and have been implicated as a putative therapeutic target cell population of anti-viral treatment. In addition, the failure of treated animals to reconstitute following lethal irradiation suggests that an immunotherapy-induced alteration occurs in the bone marrow of AKR mice.     

SEQUENTIAL OBSERVATIONS OF THYMIC LYMPHOMA DEVELOPMENT INDUCED IN 10-WEEK-OLD F344 RATS BY N-PROPYL-N-NITROSOUREA

N-Propyl-N-nitrosourea (PNU) is a strong carcinogen which induces thymic lymphoma and other tumors in F344 rats. In the present experiment, sequential changes of the thymus, spleen, bone marrow, and other organs were examined histologically in F344 rats which were continuously given PNU in the drinking water. Hematopoietic organs, such as the bone marrow, spleen, and thymus rapidly became hypoplastic. Atrophy of the thymus was followed by repopulation and hyperplasia. The latter was characterized by lymphoblast-like cells and was followed by development of early stage lymphoma, which arose from the 10th experimental week onwards. At first, thymic lymphoma was observed to involve thymic lobes unilaterally, later spreading to bilateral lobes, and at the 16th experimental week, metastatic foci were evident in the bone marrow of one rat. In contrast, hypoplasia of the bone marrow continued until the end of the experiment, while hypoplasia of the red splenic pulp continued until the 12th experimental week. These results indicate that PNU-induced lymphomas arise from within the thymus, although it is not possible to rule out a role for the bone marrow as including target cells of PNU. This PNU-thymic lymphoma system in F344 rats should serve as a good model for the study of experimentally induced thymic lymphoma.     

Early decline of thymic effect on T cell differentiation.

Thymic lobes of B6C3F1 mice ranging in age from 1 day to 11 weeks were implanted under the kidney capsule of T cell deprived syngeneic young adult TXB mice, and the capacity of the thymus grafts to influence the maturation of T cells was assessed at 6 and 12 weeks after the implantation in terms of (a) regenerative activities of the grafted thymus, (2) splenic T cell dependent anti-SRBC response, and (c) mitogenic reactivity of spleen and lymph node cells to T cell specific mitogens. The results revealed that: (1) thymic tissues from 1 week old donors were most efficient in restoring the immune potential of adult TXB mice; (2) a decline in mitogenic reactivities of spleen and lymph node cells was observed in recipients of thymus grafts from donors of 1 month and older; and a decline of splenic helper T cell function was observed in recipients of thymus grafts from 11 weeks old donors. The significance of this early decline in the thymic effect on T cell differentiation is discussed.     

The ontogeny of B cells in the thymus of normal, CD3 epsilon knockout (KO), RAG-2 KO and IL-7 transgenic mice.

The ontogeny of thymic B cells was determined by three-color flow cytometry and the presence or absence of B cell progenitors confirmed by cell culture experiments. In the thymus of young normal mice, CD117(+), B220(low) pro- and pre-B cells are present but disappear with age. B220(low), CD5(+), B-1 B cells are present in the thymus of older animals following the appearance of similar cells in the peritoneal cavity and blood. In CD3 epsilon gene-deleted mice, the phenotypic progression and number of thymic B cells remains unaltered, showing that blocking T cell development does not automatically result in an increase of thymic B lymphopoiesis. Pro-B cells in RAG-2 knockout mice are found in the fetal and neonatal blood, spleen and thymus, but with increasing age are only found in the bone marrow. B lymphopoiesis in adult IL-7 transgenic mice is dramatically altered with CD117(+) pro- and pre-B cells present in spleen, lymph node and blood. In the thymus of adult IL-7 transgenic mice, the fraction of CD117(+) thymic B cells is significantly increased. These results show that in the steady state, the phenotype of thymic B cells is critically dependent on both mouse age and the phenotype of circulating B cells.