Loya Abel

Age-related changes in the capacity of bone marrow cells to differentiate in thymic organ cultures

The capacity of the bone marrow to give rise to T cells in advanced age was studied in vitro by reconstituting fetal thymic lobes from 14-day C57BL/Ka (Thy-1.1) mice with bone marrow cells from old (24-month) or young (3-month) C57BL/6 (Thy-1.2) mice. The use of these congenic strains enabled distinguishing between donor and host contribution to the developing T cells. We found that bone marrow cells from aged mice maintained their capacity to reconstitute fetal thymic explants and to differentiate into various T-cell subsets as assessed by distinct T-cell-specific surface markers (Thy-1, Lyt-1, Lyt-2, and L3T4) and functions (concanavalin A-induced proliferative and cytotoxic responses). However, when mixtures of old and young bone marrow cells reconstituted fetal thymic explants, the cells of old mice were less efficient than those of young in their capacity to give rise to T cells. These results indicate that bone marrow cells from aged mice can reconstitute the thymus and differentiate into T cells; however, their reconstituting capacity is inferior to that of bone marrow cells from young mice.

Efficacy of anti-influenza peptide vaccine in aged mice

Influenza infections may cause serious morbidity, as well as mortality in the elderly. In the present study we vaccinated old and young mice of two strains with three synthetic recombinant constructs (Levi and Arnon, 1995. In: Chanock, R.M. et al. (Eds.), Vaccines 95. CSHL Press, New York, pp. 311-316) and examined their capacity to eliminate a challenge of virus. Virus clearance from the lungs in the aged was very efficient, although the immune response in the aged was comparatively reduced. The data demonstrate that an intranasal administration of peptide-based anti-influenza vaccine without any adjuvant can be efficient and protective in old mice. Further studies are needed to determine whether such constructs will provide an effective vaccine for elderly human subjects.

Ontogeny of T cells: development of pre-T cells from fetal liver and yolk sac in the thymus microenvironment

The patterns of development of T cells from the very early stem cells that settle in the embryonic thymus have been studied. For this purpose, mouse embryonic thymuses (14 days) depleted of thymocytes were reconstituted with hemopoietic stem cells from fetal liver (FL) and yolk sac (YS) and T-cell development was followed in vitro in organ culture. It was found that cells derived from FL and YS of 10- to 14-day-old embryos were capable of reconstituting depleted thymic explants and exhibiting membrane markers in a pattern similar to that of thymocytes developing in intact thymic explants. Furthermore, these cells responded to concanavalin A in proliferative and cytotoxic assays as measured by limiting-dilution analysis. Thus, lymphohemopoietic stem cells emerging in the embryo prior to thymus lymphoid development are capable of differentiation in the thymus microenvironment into T cells, identified by phenotypic markers and functions that are characteristic of cells developing in the intact embryonic thymus.

Quantitative analysis of bone marrow thymic progenitors in young and aged mice

Our studies on the capacity of bone marrow (BM) to generate T lymphocytes in aging have revealed that under the competitive conditions of thymic reconstitution, cells of aged mice are significantly inferior to those of the young. The present study was designed to further investigate the basis of this age-related change. Two mechanisms were considered: (a) The potential of BM-derived T cell precursors from aged mice to proliferate and differentiate in the thymic microenvironment is impaired. (b) The frequency of T cell precursors is reduced in BM of aged mice, thus affecting their ability to compete efficiently in reconstituting the thymus. These possibilities were studied in vitro by colonizing thymocyte-depleted fetal thymic lobes with BM cells from aged (24-month) and young (3-month) C57BL/6 mice. By determining the cell cycle duration of BM-derived cells which have seeded the thymic lobes, we found that cells originating from aged mice proliferate in the thymus at the same rate as those from young mice. Reconstitution with limiting numbers of BM cells indicated that the frequency of thymic progenitors in the BM is significantly reduced in aged as compared to young mice. We thus conclude that aging is associated with a quantitative reduction in the frequency of thymic progenitors in the BM.

Thymocyte progenitors and T cell development in aging

Dysfunction of T lymphocytes in aging has been causally related to a gradual loss of the thymic microenvironmental function. However, in view of the fact that T cells are generated from bone marrow-derived stem cells that settle in the thymus, we have investigated the possibility that aging effects on the bone marrow have an impact on T cell development. Our approach was based on seeding of bone marrow cells, from young and old mice, onto lymphoid-depleted fetal thymus explants, and examining the patterns of T lymphocyte development under organ culture conditions. The results indicate multifactorial effects of aging, on pre-thymic and intra-thymic development processes, as well as on feedback regulation by mature T cells.

A mathematical model of the effect of aging on bone marrow cells colonizing the thymus

The process of T cell generation in the thymus involves complex cell-cell interactions between the various types of thymic stromal cells, thymocyte progenitors, thymocytes at different stages of differentiation and external factors. We applied the tool of mathematical modelling to analyze hypotheses and direct experiments concerning mechanisms underlying the observed developmental inferiority of bone-marrow thymocyte progenitors from old mice. Previous experimental data showed that lower cell numbers were obtained from old bone marrow-derived thymocyte progenitors, compared to young bone marrow-derived progenitors, when colonizing simultaneously the same fetal thymus. In this study, simulations based on the mathematical model indicate that the developmental inferiority of old bone marrow-derived progenitors cannot be explained by a change in a single parameter, such as the observed differences in progenitor frequency, an increase in cell cycle duration, a reduction in the fraction of proliferating cells in old age, and/or an increase in the rate of cell death. We have performed experimental measurements of the fractions of cycling cells. No significant difference was found between these fractions in young and old bone marrow-derived thymocytes. The difference in developmental patterns of young and old bone marrow-derived thymocytes may be due to a combination of more than one mechanism, possibly including interactions between competing thymocytes of old and young bone marrow origin.

Developmental interactions of CD4 T cells and thymocytes: age-related differential effects

The study was designed to determine whether the developmental potential of immature thymocytes in the thymus is altered in aging, and whether concomitantly present mature T cells have any feedback effect. The strategy was to seed sorted double negative, CD4-CD8-(DN) thymocytes on their own, or in the presence of mature T cells, onto lymphoid depleted fetal thymus (FT) explants, and to examine the resulting T cell subsets. Thymocyte donors were young (2-3 months) and old (24 months) C57BL/6J, Thy1.2 mice and splenocytes were from C57BL/Ka, Thy1.1 mice. The DN cells of the old gave rise to lower values of double positive CD4+CD8+ (DP) cells than those of the young. Cocultures containing a mixture of DN thymocytes and CD4+CD8- splenocytes showed higher CD4+CD8- and DN, and lower DP and CD4-CD8+ levels in the old-donor derived cells, as compared with the young ones. Similar results were obtained with CD4+CD8- thymocytes. In contrast, the presence of CD4-CD8+ splenocytes had no effect on the pattern of DN cell development. Our data indicate that differentiation of CD4/CD8 thymocyte phenotypes is affected by CD4+ cells, in an age-associated differential manner.

Checkpoints in thymocytopoiesis in aging: expression of the recombination activating genes RAG-1 and RAG-2

The study was designed to establish whether the ability to rearrange the T cell receptor (TCR) Vbeta genes is altered with age. We examined the expression of recombinase activating genes, RAG-1 and RAG-2, in the thymus of mice at different ages (2-24 months). A significant age-related decrease in RAG-1 and RAG-2 expression was observed in the thymocytes from the age of 12 months and over. To find out if this decrease is determined in the thymocyte progenitors or induced by the thymic microenvironment, we co-cultured lymphoid depleted fetal thymus (FT) explants with bone marrow cells, or immature thymocytes, from young and old mice. The developing thymocytes were examined at different time intervals during the first week of culture. Whereas cells derived from the immature thymocytes of the old donors failed to express RAG-1 and RAG-2, compared to the young, the bone marrow derived cells of both age groups did show this expression, and there was no difference in Vbeta rearrangement of the TCR. Our study indicates that T cell progenitors in the aging bone marrow retain the potential to give rise to T cells with TCR rearrangements, and the expression is determined by the thymic stroma.

Aging in the T Lymphocyte Compartment

A decline in the capacity of bone marrow cells to differentiate to T lymphocytes was found when cells from young and old donors were seeded onto an alymphoid fetal thymus. A step-by-step analysis of cell-cell interactions of the lymphohemopoietic cells and the thymic stroma indicated an effect of age on a variety of cell differentiation parameters. These included a decrease in the affinity of bone marrow cells to the stroma, and in their capacity to compete with the thymic lymphoid resident cells on colonization of the thymus. There was a significant decrease in the ability of cells of old donors to replicate sequentially within the thymic microenvironment. There was a reduced capacity of bone marrow cells from aging mice to express a developmental preference after seeding onto a syngeneic fetal thymus in a mixture with cells from allogeneic donors. We addressed the question whether the aging thymus contains increased levels of immature cells that fail to differentiate in the involuted thymic microenvironment by seeding thymocytes from young and old donors onto the fetal thymic stroma. The values of T cells that developed from the old donor inoculum were lower under these conditions. Our studies suggest that at least some of the manifestations of aging in the T cell compartment are related to developmentally programmed events in the lymphohemopoietic cell compartment.

Immune reactivity during ageing III. Removal of peanut-agglutinin binding cells from ageing mouse spleen cells leads to increased reactivity to mitogens

Spleen cells from ageing (24--30-month-old) mice, manifesting low response to concanavalin A (Con A) and phytohemagglutinin (PHA), were separated by peanut agglutinin (PNA) into agglutinable (PNA+) and unagglutinable (PNA-) fractions. The PNA+ cells were found to suppress the response of young (2--3-month-old) mouse spleen cells to the mitogens Con A and PHA. On the other hand, PNA- cells of the ageing mice expressed a response to these mitogens, at levels higher than those of the unseparated cells. Re-mixing of the PNA- and PNA+ cells of the ageing mouse spleens at various proportions indicated that the PNA- cells are indeed suppressible by the PNA+ cells. Anti Thy-1.2 treatment to the PNA+ fraction suggested that this suppression was not exerted by T cells. It thus appears that at least part of the reduced lymphoid cell function in ageing is related to an effect of suppressor cells, interfering with the expression of available reactive cells.