[Frontiers in Bioscience 3, d59-99, January 15, 1998]

	[Frontiers in Bioscience 3, d59-99, January 15, 1998] Reprints PubMed CAVEAT LECTOR
	T CELLS AND AGING Graham Pawelec ¹, Ed Remarque ², Yvonne Barnett ³, Rafael Solana⁴ 1 University of Tübingen, Tübingen, FRG ²,University of Leiden, Leiden, Holland ³, University of Ulster, Coleraine, Northern Ireland ⁴, University of Córdoba, Córdoba, Spain Received 12/29/97 Accepted 1/5/97 4. POSSIBLE CAUSES OF IMMUNOSENESCENCE 4.1 Hematopoiesis Dysregulated hematopoiesis is seen in elderly individuals, raising the possibility that multiple lesions are responsible for altered immune function in the aged. Hematopoiesis may be compromized because of a severely reduced capacity to produce granulocyte/macrophage colony stimulating factor (GM-CSF) (28) and because lower numbers of progenitor cells are present in the BM (29). Normal cells with shorter telomeres possess less remaining replicative capacity than those with longer telomeres (see section 7). Accordingly, stem cells from adult BM have shorter telomeres than fetal liver-derived or umbilical cord-derived stem cells, and telomere lengths decrease on culture (30). This occurs despite low level expression of telomerase in these cells (31), although the rate of base-pair loss per population doubling of cells in culture is lower during the first two weeks, when telomerase is upregulated, than in the next two, when it is downregulated (32). The telomerase expressed is therefore functionally relevant. A survey of 500 autotransplant candidates (33) concluded that aging was associated with reduced numbers of committed hematopoietic progenitor cells, as measured by surface phenotype (CD34⁺, Thy1⁺, CD38^lo) and function (long-term culture initiation). In mice, the repopulating potential of murine fetal liver-derived stem cells is higher than that of adult BM-derived stem cells (34). Together, these sporadic results may suggest compromized ability to generate progenitor cells from BM in the elderly. Of direct clinical significance is the recent evidence that CD34⁺ cells mobilize less effectively in cytokine-treated elderly compared to young donors (35). Moreover, the capacity of progenitor T cells from old BM to develop in the thymus may also be compromized (36). However, this has not been found in all models (37) where young and old BM was identical in reconstitution ability but age of the thymic stroma was found to be critical for the development of autoimmunity. The reasons for these differences are presently unclear. By studying thymocytes generated in vitro from young and old donor-derived progenitor cells, co-cultured in the presence of lymphoid-depleted fetal thymus, decreased generation of CD4/8-double negative thymocyte progenitors was demonstrated, along with a developmental arrest at the transition from CD44+ CD4/8-double negative to CD44-negative, CD4/8 double positive cells. This does suggest an intrinsic change in the stem cells with age (38). Other studies in mice have found that hematopoietic stem cells are more frequent in old individuals and more likely to be in cycle, although they were less efficient at homing to and engrafting bone marrow of irradiated recipients (39). Some of the previously published inconsistencies in the data may be resolved by the study of de Haan et al . (40). These investigators showed that aging significantly alters the primitive hematopoietic compartments of mice in several ways: firstly, the proliferative activity of the primitive cells is greatly reduced over the first year of life; secondly, there is a (compensatory) increase in relative and absolute stem cell number with age; thirdly, the changes are strain-dependent and related both to the longevity of the strain as well as to the age of the individual mouse. A strong inverse correlation was observed between mouse lifespan and the number of autonomously cycling progenitors in 8 different strains of mice; a gene controlling this frequency was mapped to mouse chromosome number 18 (syntenic to human chr. no. 5, involved with various haematological malignancies, ref. 41). In outbred species such as humans, this type of variation would make analysis difficult. Therefore, more work needs to be done to definitively answer the question of whether any alterations in hematopoiesis in the elderly may contribute to immunosenescence. 4.2 Thymus It is accepted that T cell differentiation is compromized with age because of thymic involution. However, even this generality may not be universally and incontrovertably true. Thus, a recent study of thymic samples from donors from one week to 50 years old showed an early decrease of cellularity but with two early peaks at 9 months and 10 years of age. Moreover, the adult thymus still contained thymocytes with similar surface phenotypes to those seen in young donors (42). This suggests that the thymus can remain active at leat up to middle age, but the functional activities of the thymus output could not be studied in this investigation. There may be a strong genetic contribution influencing thymic involution; for example, rats of the Buffalo strain do not experience thymic involution and in parallel do not manifest decreased T cell function with age (43). Again, therefore, genetic heterogeneity in outbred populations might be expected to contribute to marked inter-individual differences. Thymic involution may itself be affected by the status of the T cells in the individual; for example, thymic involution is reported not to occur in T cell antigen receptor (TCR)-transgenic mice, leading to the conclusion that successfully matured T cells can maintain thymic integrity (44,45). In addition, reconstitution experiments indicate that the observed accelerated maturation of T cells to a memory phenotype in old mice is due to the aged environment and involves interactions via the TCR which are, however, not antigen-specific [(46) and M. Thoman, cited in ref. (1)]. T cells also affect the thymus itself via a feedback effect and provide survival signals for the medullary microenvironment. An important survival signal of this type may be IL 4 [M. Ritter, cited in (1)]. Thus, the aging of stem cells and/or T cell precursors may directly influence processes of thymic involution. CD4 T cells appear to be the most effective at maintaining thymic function and a decreased collaboration between thymocyte progenitors and mature CD4+ T cells from aged mice could also result in a defective feedback of aged CD4+ cells on thymocyte development and differentiation [(47) and A. Globerson, cited in (1)]. Signals controlling thymic status may also be derived from the nervous system, either directly from sympathetic innervation or indirectly via the hypothalamic-pituitary axis (48). There are increased numbers of noradrenergic sympathetic nerves and 15-fold increases in concentration of norepinefrine in the thymi of 24 month-old mice (49). The phenomenon of thymic involution begins very early in life, even before puberty, and progressively continues. However, despite the assumption that thymic involution is essentially complete in adulthood, there are data to suggest that the replacement of thymic parenchyma with adipose tissue is a discontinuous process, reaching a maximum at around 50 years of age in humans and thereafter not progressing further (50). Moreover, the amount of non-fatty material in the thymus may not decrease further after the age of about 30 years (50). Secretion of the important immunoactive hormone, thymulin, continues throughout life, although blood thymulin levels do decrease with age (51). There is evidence here, however, that lower levels of thyroid hormones and insulin, rather than thymus dysfunction, are responsible for lower thymulin levels (51). These findings, together with the genetic heterogeneity of outbred populations probably influencing the occurrence and rate of thymic involution, make it difficult to assess the contribution of such involution to changes in T cell function in man. There is evidence to suggest that even in (some of) the very old, sufficient thymic function may be retained to allow for naive T cell differentiation (52). It has been estimated that complete thymic atrophy in humans would not occur until the age of about 120 years (53). The decrease in thymic size and alterations in thymic architecture and functionality for T cell differentiation which do occur up to middle age are the results of a controlled process independent of stress and lack of repair mechanisms. Thus, infection, pregnancy, stress, drug or hibernation-induced thymic involution are all reversible in younger individuals, leading to the suggestion that thymic atrophy is an energy-saving process according to the disposable soma theory of aging (54). According to this view, the evolutionary pressures on maintaining thymic function for constant full T cell repertoire generation were secondary to the generation early in life of a memory cell repertoire for a mostly tribally-limited pathogen presence. Thymic function did not need to be maintained beyond reproductive maturity because the number of new infections experienced by early humans in later life in the wild was too limited to make thymic maintenance worthwhile. This presupposes that early humans did not come into contact with very many new pathogens, suggesting a sedentary existence. However, early humans were nomadic, only recently becoming sedentary, so it is unclear whether this does apply. George & Ritter suggested (54) correlating thymic involution rates and function in animals and birds which migrate long distances, the hypothesis being that the more varied the environment, the more evolutionary pressure there would be to maintain the thymus. Another possibility to explain early thymic involution may relate to avoidance of undesired tolerization of newly generated T cells to pathogens which in later life have entered the thymus (55). Whatever the reason, the effects of thymic changes, associated with increased age, on the immune system in mice are marked. As mentioned above, the situation in humans may not be so clear. Moreover, in mice, age-related changes in the thymus may influence other organ systems in some manner, as has been reported for effects on the liver (56). In mouse, the number of T cells exported from the thymus decreases with age, as does the ability of thymic epithelium from old animals to support the differentiation of T cells from young animals´ BM. The type of T cell produced is affected by aging. There may be a developmental block which results in an increase in the frequency of CD3⁺ CD4/8-DN thymocytes (57). This may result in higher proportions of apparently immature T cells being present in old individuals (consistent with decreased thymic function; refs. 58-60). However, the markers used to discriminate immature T cells in these latter studies do not seem to have allowed for distinguishing between CD2⁺ T cells and CD2⁺NK cells, so that the increase in immature T cells might actually represent an increase in NK cells. One group specifically tested this and concluded that such cells were indeed functionally active NK cells (61). Other important changes related to altered thymic function may include changed restriction repertoires of the T cells generated, such that even TCR2 (TCR-a b ) cells acquire responsiveness to antigen presented by non-self MHC in man (27) and mouse (26). Evidence has also been presented for increased levels of extra-thymically-differentiated T cells in elderly humans, as well as increased NK-phenotype (but not NK-functional) cells (62). The increase in extra-thymically-differentiated T cells may also represent some sort of compensatory mechanism for decreased thymic integrity. Studies on depletion of CD4⁺ cells by CD4 mAb indicate that recovery of this population, which is dependent upon the presence of the thymus, is much slower in aged mice than young mice (63). In humans, CD4-depletion by mAb treatment in rheumatoid arthritis (RA) results in a very prolonged effect. T cell reconstitution is slow, there is a predominance of T cells with memory phenotypes, and there is limited TCR diversity (64). The ability to generate new T lymphocytes after chemotherapy is inversely related to the patients´ age, probably an indirect indication of thymic involution (65). During the first year of recovery after chemotherapy, the CD4 cells in adults also mostly carry memory markers, but in children they carry markers of naive T cell (66). Recovery of CD4 cells was inversely related to age of donor and was enhanced in patients with thymic enlargement after chemotherapy (67). However, CD8 cell recovery was much more rapid and was not associated with age or thymic enlargement. The CD8 cells were mostly CD57+ CD28-negative. All these data can be interpreted to imply that the prime source of reconstituting cells in adults is from peripheral expansion of pre-existing CD4 T cell subsets which survived conditioning, and not by thymus-dependent generation of new T cells. CD8 cell generation is thought to be extrathymic here (67). A similar phenomenon may be observed in HIV infection, where antiviral therapy results in an increase of naive CD4 cells only if some were still present before initiation of therapy (68). It is noteworthy that in bone marrow transplant (BMT) patients, even as long as 5 years after transplantation, CD4 cell counts are still depressed and cells with a naive phenotype are also rare. Cells with a memory phenotype (CD45RA^lo, CD29^hi, CD11a^hi) were abundant and many of these were CD28-negative. Moreover, there was a negative correlation between the ability to produce naive T cells after BMT and patient age (69), independently of the presence of graft-versus-host disease. These findings seem to apply only to naive T cells, as might be expected; thus, Koehne et al . reported that in human peripheral blood stem cell transplantation, only recovery of the CD4⁺CD45RA⁺ population, but not the CD45RO⁺ population, was thymus-dependent (70). A bone marrow-transplanted young thymectomized patient mimicked this phentoype (71). The patient showed preferential recovery of CD45RO+ cells in the CD4 subset, although in CD8 cells, CD45RA+ cells were generated as well as in age-matchd euthymic patients. It was therefore concluded that a functional thymus was essential for the generation of naive CD4 cells, although extrathymic pathways for naive CD8 cell generation appeared functional (71). Following T cell-depleted BMT, loss of TCR diversity in slowly reconstituting cells is also seen, again consistent with peripheral expansion of a very limited number of T cells transferred with the graft (72). 4.3 Post-thymic aging Finally, mature T cells are also subject to aging processes, either of the type affecting post-mitotic cells (when quiescent) or of the "replicative senescence" type (during clonal expansion for effective immune responses). This will be discussed at length in the following sections.

[Frontiers in Bioscience 3, d59-99, January 15, 1998]
Reprints
PubMed
CAVEAT LECTOR

T CELLS AND AGING

Graham Pawelec ¹, Ed Remarque ², Yvonne Barnett ³, Rafael Solana⁴

1 University of Tübingen, Tübingen, FRG ²,University of Leiden, Leiden, Holland ³, University of Ulster, Coleraine, Northern Ireland ⁴, University of Córdoba, Córdoba, Spain

Received 12/29/97 Accepted 1/5/97

4. POSSIBLE CAUSES OF IMMUNOSENESCENCE

4.1 Hematopoiesis

Dysregulated hematopoiesis is seen in elderly individuals, raising the possibility that multiple lesions are responsible for altered immune function in the aged. Hematopoiesis may be compromized because of a severely reduced capacity to produce granulocyte/macrophage colony stimulating factor (GM-CSF) (28) and because lower numbers of progenitor cells are present in the BM (29). Normal cells with shorter telomeres possess less remaining replicative capacity than those with longer telomeres (see section 7). Accordingly, stem cells from adult BM have shorter telomeres than fetal liver-derived or umbilical cord-derived stem cells, and telomere lengths decrease on culture (30). This occurs despite low level expression of telomerase in these cells (31), although the rate of base-pair loss per population doubling of cells in culture is lower during the first two weeks, when telomerase is upregulated, than in the next two, when it is downregulated (32). The telomerase expressed is therefore functionally relevant. A survey of 500 autotransplant candidates (33) concluded that aging was associated with reduced numbers of committed hematopoietic progenitor cells, as measured by surface phenotype (CD34⁺, Thy1⁺, CD38^lo) and function (long-term culture initiation). In mice, the repopulating potential of murine fetal liver-derived stem cells is higher than that of adult BM-derived stem cells (34). Together, these sporadic results may suggest compromized ability to generate progenitor cells from BM in the elderly. Of direct clinical significance is the recent evidence that CD34⁺ cells mobilize less effectively in cytokine-treated elderly compared to young donors (35). Moreover, the capacity of progenitor T cells from old BM to develop in the thymus may also be compromized (36). However, this has not been found in all models (37) where young and old BM was identical in reconstitution ability but age of the thymic stroma was found to be critical for the development of autoimmunity. The reasons for these differences are presently unclear. By studying thymocytes generated in vitro from young and old donor-derived progenitor cells, co-cultured in the presence of lymphoid-depleted fetal thymus, decreased generation of CD4/8-double negative thymocyte progenitors was demonstrated, along with a developmental arrest at the transition from CD44+ CD4/8-double negative to CD44-negative, CD4/8 double positive cells. This does suggest an intrinsic change in the stem cells with age (38).

Other studies in mice have found that hematopoietic stem cells are more frequent in old individuals and more likely to be in cycle, although they were less efficient at homing to and engrafting bone marrow of irradiated recipients (39). Some of the previously published inconsistencies in the data may be resolved by the study of de Haan et al . (40). These investigators showed that aging significantly alters the primitive hematopoietic compartments of mice in several ways: firstly, the proliferative activity of the primitive cells is greatly reduced over the first year of life; secondly, there is a (compensatory) increase in relative and absolute stem cell number with age; thirdly, the changes are strain-dependent and related both to the longevity of the strain as well as to the age of the individual mouse. A strong inverse correlation was observed between mouse lifespan and the number of autonomously cycling progenitors in 8 different strains of mice; a gene controlling this frequency was mapped to mouse chromosome number 18 (syntenic to human chr. no. 5, involved with various haematological malignancies, ref. 41). In outbred species such as humans, this type of variation would make analysis difficult. Therefore, more work needs to be done to definitively answer the question of whether any alterations in hematopoiesis in the elderly may contribute to immunosenescence.

4.2 Thymus

It is accepted that T cell differentiation is compromized with age because of thymic involution. However, even this generality may not be universally and incontrovertably true. Thus, a recent study of thymic samples from donors from one week to 50 years old showed an early decrease of cellularity but with two early peaks at 9 months and 10 years of age. Moreover, the adult thymus still contained thymocytes with similar surface phenotypes to those seen in young donors (42). This suggests that the thymus can remain active at leat up to middle age, but the functional activities of the thymus output could not be studied in this investigation. There may be a strong genetic contribution influencing thymic involution; for example, rats of the Buffalo strain do not experience thymic involution and in parallel do not manifest decreased T cell function with age (43). Again, therefore, genetic heterogeneity in outbred populations might be expected to contribute to marked inter-individual differences. Thymic involution may itself be affected by the status of the T cells in the individual; for example, thymic involution is reported not to occur in T cell antigen receptor (TCR)-transgenic mice, leading to the conclusion that successfully matured T cells can maintain thymic integrity (44,45). In addition, reconstitution experiments indicate that the observed accelerated maturation of T cells to a memory phenotype in old mice is due to the aged environment and involves interactions via the TCR which are, however, not antigen-specific [(46) and M. Thoman, cited in ref. (1)]. T cells also affect the thymus itself via a feedback effect and provide survival signals for the medullary microenvironment. An important survival signal of this type may be IL 4 [M. Ritter, cited in (1)]. Thus, the aging of stem cells and/or T cell precursors may directly influence processes of thymic involution. CD4 T cells appear to be the most effective at maintaining thymic function and a decreased collaboration between thymocyte progenitors and mature CD4+ T cells from aged mice could also result in a defective feedback of aged CD4+ cells on thymocyte development and differentiation [(47) and A. Globerson, cited in (1)]. Signals controlling thymic status may also be derived from the nervous system, either directly from sympathetic innervation or indirectly via the hypothalamic-pituitary axis (48). There are increased numbers of noradrenergic sympathetic nerves and 15-fold increases in concentration of norepinefrine in the thymi of 24 month-old mice (49).

The phenomenon of thymic involution begins very early in life, even before puberty, and progressively continues. However, despite the assumption that thymic involution is essentially complete in adulthood, there are data to suggest that the replacement of thymic parenchyma with adipose tissue is a discontinuous process, reaching a maximum at around 50 years of age in humans and thereafter not progressing further (50). Moreover, the amount of non-fatty material in the thymus may not decrease further after the age of about 30 years (50). Secretion of the important immunoactive hormone, thymulin, continues throughout life, although blood thymulin levels do decrease with age (51). There is evidence here, however, that lower levels of thyroid hormones and insulin, rather than thymus dysfunction, are responsible for lower thymulin levels (51). These findings, together with the genetic heterogeneity of outbred populations probably influencing the occurrence and rate of thymic involution, make it difficult to assess the contribution of such involution to changes in T cell function in man. There is evidence to suggest that even in (some of) the very old, sufficient thymic function may be retained to allow for naive T cell differentiation (52). It has been estimated that complete thymic atrophy in humans would not occur until the age of about 120 years (53). The decrease in thymic size and alterations in thymic architecture and functionality for T cell differentiation which do occur up to middle age are the results of a controlled process independent of stress and lack of repair mechanisms. Thus, infection, pregnancy, stress, drug or hibernation-induced thymic involution are all reversible in younger individuals, leading to the suggestion that thymic atrophy is an energy-saving process according to the disposable soma theory of aging (54). According to this view, the evolutionary pressures on maintaining thymic function for constant full T cell repertoire generation were secondary to the generation early in life of a memory cell repertoire for a mostly tribally-limited pathogen presence. Thymic function did not need to be maintained beyond reproductive maturity because the number of new infections experienced by early humans in later life in the wild was too limited to make thymic maintenance worthwhile. This presupposes that early humans did not come into contact with very many new pathogens, suggesting a sedentary existence. However, early humans were nomadic, only recently becoming sedentary, so it is unclear whether this does apply. George & Ritter suggested (54) correlating thymic involution rates and function in animals and birds which migrate long distances, the hypothesis being that the more varied the environment, the more evolutionary pressure there would be to maintain the thymus. Another possibility to explain early thymic involution may relate to avoidance of undesired tolerization of newly generated T cells to pathogens which in later life have entered the thymus (55).

Whatever the reason, the effects of thymic changes, associated with increased age, on the immune system in mice are marked. As mentioned above, the situation in humans may not be so clear. Moreover, in mice, age-related changes in the thymus may influence other organ systems in some manner, as has been reported for effects on the liver (56). In mouse, the number of T cells exported from the thymus decreases with age, as does the ability of thymic epithelium from old animals to support the differentiation of T cells from young animals´ BM. The type of T cell produced is affected by aging. There may be a developmental block which results in an increase in the frequency of CD3⁺ CD4/8-DN thymocytes (57). This may result in higher proportions of apparently immature T cells being present in old individuals (consistent with decreased thymic function; refs. 58-60). However, the markers used to discriminate immature T cells in these latter studies do not seem to have allowed for distinguishing between CD2⁺ T cells and CD2⁺NK cells, so that the increase in immature T cells might actually represent an increase in NK cells. One group specifically tested this and concluded that such cells were indeed functionally active NK cells (61). Other important changes related to altered thymic function may include changed restriction repertoires of the T cells generated, such that even TCR2 (TCR-a b ) cells acquire responsiveness to antigen presented by non-self MHC in man (27) and mouse (26). Evidence has also been presented for increased levels of extra-thymically-differentiated T cells in elderly humans, as well as increased NK-phenotype (but not NK-functional) cells (62). The increase in extra-thymically-differentiated T cells may also represent some sort of compensatory mechanism for decreased thymic integrity.

Studies on depletion of CD4⁺ cells by CD4 mAb indicate that recovery of this population, which is dependent upon the presence of the thymus, is much slower in aged mice than young mice (63). In humans, CD4-depletion by mAb treatment in rheumatoid arthritis (RA) results in a very prolonged effect. T cell reconstitution is slow, there is a predominance of T cells with memory phenotypes, and there is limited TCR diversity (64). The ability to generate new T lymphocytes after chemotherapy is inversely related to the patients´ age, probably an indirect indication of thymic involution (65). During the first year of recovery after chemotherapy, the CD4 cells in adults also mostly carry memory markers, but in children they carry markers of naive T cell (66). Recovery of CD4 cells was inversely related to age of donor and was enhanced in patients with thymic enlargement after chemotherapy (67). However, CD8 cell recovery was much more rapid and was not associated with age or thymic enlargement. The CD8 cells were mostly CD57+ CD28-negative. All these data can be interpreted to imply that the prime source of reconstituting cells in adults is from peripheral expansion of pre-existing CD4 T cell subsets which survived conditioning, and not by thymus-dependent generation of new T cells. CD8 cell generation is thought to be extrathymic here (67). A similar phenomenon may be observed in HIV infection, where antiviral therapy results in an increase of naive CD4 cells only if some were still present before initiation of therapy (68). It is noteworthy that in bone marrow transplant (BMT) patients, even as long as 5 years after transplantation, CD4 cell counts are still depressed and cells with a naive phenotype are also rare. Cells with a memory phenotype (CD45RA^lo, CD29^hi, CD11a^hi) were abundant and many of these were CD28-negative. Moreover, there was a negative correlation between the ability to produce naive T cells after BMT and patient age (69), independently of the presence of graft-versus-host disease. These findings seem to apply only to naive T cells, as might be expected; thus, Koehne et al . reported that in human peripheral blood stem cell transplantation, only recovery of the CD4⁺CD45RA⁺ population, but not the CD45RO⁺ population, was thymus-dependent (70). A bone marrow-transplanted young thymectomized patient mimicked this phentoype (71). The patient showed preferential recovery of CD45RO+ cells in the CD4 subset, although in CD8 cells, CD45RA+ cells were generated as well as in age-matchd euthymic patients. It was therefore concluded that a functional thymus was essential for the generation of naive CD4 cells, although extrathymic pathways for naive CD8 cell generation appeared functional (71). Following T cell-depleted BMT, loss of TCR diversity in slowly reconstituting cells is also seen, again consistent with peripheral expansion of a very limited number of T cells transferred with the graft (72).

4.3 Post-thymic aging

Finally, mature T cells are also subject to aging processes, either of the type affecting post-mitotic cells (when quiescent) or of the "replicative senescence" type (during clonal expansion for effective immune responses). This will be discussed at length in the following sections.