[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 5. MECHANISMS CONTRIBUTING TO IMMUNOSENESCENCE 5.1 Accessory cells Decreased T cell responses in the elderly may be due to decreased T cell function, decreased accessory cell function, or both. There is evidence for accessory cell changes (73) and monocytes are clearly compromized in their function in the elderly, eg. in that they secrete less IL 1 and have decreased cytotoxicity and protein kinase translocation (74). A more recent analysis suggested that lipopolysaccharide (LPS)-stimulated monocytes from the elderly produced less G-CSF, GM-CSF, IL 8, TNF-a , and MIP-1-a as well as less IL 1ß compared to those from young donors (75). In some clinically-relevant animal models, it is the accessory cells which seem to contribute critically to age-associated suboptimal responses, eg. in the response of mice to trypanosome antigens (76). Another example where T and B cell function appears to be normal, but accessory cell function is compromized in aged mice comes from a vaccination model using pneumococcal preparations (77). To distinguish between T cell and APC alterations may be difficult but can be approached in certain models by using purified T cells in the absence of accessory cells. For example, using mitogenic CD2 mAb and soluble costimulatory factors (cytokines, phorbol esthers, mAb), Beckman et al . (78) have shown that in CD45RO⁺ CD4 cells, the only pathway not comparable between young and old donors was for stimulation by CD2 in combination with IL 7. Thus, signalling may be intact in old memory cells, except for IL 7-dependent pathways. In contrast, CD45RA⁺ cells from old donors responded less well than young naive cells to CD2 + IL 2, IL 6, IL 7, IL 1 or phorbol esther, suggesting multiple deficiencies in the naive cells but not the memory cells of old donors. On the other hand, dendritic cells (DC) obtained from elderly persons are reported to be able to present antigen at least as well, if not better, than DC from young donors (79). The same group also reported that DC from the elderly were able to inhibit apoptosis and stimulate proliferation in pre-senescent cultured T cells (80). These results suggest that at least a subset of APC in the elderly retain optimal function. On the other hand, it must be borne in mind that these results were obtained using DC generated in vitro using IL 4 and GM-CSF. Since the production of GM-CSF in the elderly is decreased (see section 4.1) there may not be so many functional DC available in old donors. 5.2 Alterations in signal transduction That the earliest events in T cell activation are compromized in the elderly is reflected in findings that cell surface alterations associated with activation are affected, eg. CD69 and CD71 upregulation does not take place (81). Incomplete T cell activation may be caused in the first instance by disturbed signal transduction. Particularly in T cells there are multiple levels where lesions might be sought, since signal transduction through one, several or all of the TCR, costimulatory receptors, or growth factor receptors could be compromized with age. There is evidence for alterations in all three of these possible loci. Damage to the cytoskeleton paralleling aging may have profound effects on cell function. In the case of T cells this may be relevant even to the earliest stages of T cell activation, because, for example, the signal-transducing TCR zeta chain component is associated at the cell surface with the cytoskeleton (82). If this has something to do with receptor recycling or maintaining correct three-dimensional structure of the receptor, cytoskeletal dysfunction could have profound effects on TCR signal transduction. Certainly, initial biochemical events following TCR triggering (formation of second messengers such as IP3 and DAG) are compromized, although the activity of PLC (which is responsible for IP3 and DAG generation) appears conserved in old T cells (83). However, the actual amount of PLC present in freshly isolated cells may be decreased with aging (84). Antibody against the signal-transducing CD3 zeta chain precipitates a series of tyrosine-phosphorylated proteins, the levels of which decline with age (85). Therefore, the level of expression of the TCR components and/or their ability to transduce signals may be compromized in old T cells. Signalling pathways mediated by the family of mitogen-activated protein kinases (MAPK) are considered essential for normal cell growth and function; CD3 stimulated human T cells from 50% of old subjects were found to show reductions in MAPK activation. Stimulation with phorbol esther in combination with calcium ionophore resulted in greater MAPK activation in old cells, but still not to the same extent as young cells (86), suggesting signalling deficits between the TCR and the inducers of MAPK. MAPK are activated by another protein kinase, MEK; in mouse CD4 cells, there is an age-associated decrease in MEK (87). Moreover, kinases are commonly counter-regulated by phosphatases, and even if kinase decrease were not to occur, increase in phosphatase activity might have the same result. In the case of MAPK, it has been reported that expression of MAP phosphatase is indeed increased (in aged rat hepatocytes, at least) (88). In T cells, signalling through the TCR, CD4, CD8 or the IL 2R resulted in lowered protein tyrosine kinase activity in cells from old compared to young donors, although direct activation of protein tyrosine kinases (PTK) by pervanadate was normal in the old (89). It is therefore not yet clear whether the age-related decreased tyrosine phosphorlylation observed in CD3-stimulated human T cells is related to changes in PTKs or phosphatases (PTPases). Recent data from Whisler et al . indicate that CD45-PTPase activity in old cells after CD3-stimulation is not increased compared to young cells (90). They further found that fyn enzymatic activity but not lck activity was reduced in a high proportion of T cells from the elderly compared to the young, although protein levels were the same. They concluded that decreased fyn activation but not increased PTPase activity may contribute to lowered responses in the elderly (90). Cell cycle analyses of PHA-stimulated cells from aged donors indicate a decreased frequency of cells entering S-phase with this age-related impairment of G1 progression correlating with decreased expression of c-jun, c-myc, c-myb, IL 2 and CD25 (91-93). The proportion of cells expressing c-myc (G0 to G1 marker) and c-myb (G1 to S marker) was decreased after PHA stimulation of old T cells, but the amount per cell seemed to remain the same as in young T cells (93). T cells retaining antigen recognition and effector function, yet apparently in a post-mitotic senescent or pre-senescent state have been described (94). These investigators also demonstrated that aged human T cells paralleled the senescent phenotype of fibroblasts in that on restimulation, fewer cells responded by entering the cell cycle, the remainder being arrested before S-phase. The cell cycle was also prolonged in those ca. 20% of senescent cells which could be restimulated (95). At least some of these results may reflect the situation in vivo, where PHA stimulation resulted in an earlier accumulation of cells in S phase in young donors´ T cells, and a significant delay, but eventually equivalent level, of S phase cells in the elderly (96). In Fischer rats, the age-associated decrease in IL 2 mRNA and protein correlates with a decreasing ability of nuclear extracts of freshly isolated T cells to bind an oligonucleotide representing the transcription factor NF-AT (97), suggesting differences in transcriptional regulation in young and old cells. NF-AT forms an important family of at least four transcription factors; NF-AT DNA binding activity has been found in nuclear extracts of stimulated T cells (98) and is thought to be important for IL 2 gene transcription (99). A set of transcription factors involving complexes of the various c-jun and c-fos proteins is involved in regulating transcription of many genes, including IL 2, and activation of AP-1 is detected a few hours after T cell stimulation (100,101). Specific defects in AP-1 activation have been reported in young T cell clones rendered anergic in vitro (102). The anergic phenotype is in some ways similar to the senescent phenotype (ie. cells can be stimulated via the TCR to secrete cytokines, be cytotoxic, but they cannot expand clonally via autocrine IL 2 production). AP-1 activation may be impaired in in vivo-aged human T cells as well (103). Using T cells from elderly donors rigorously selected for good health according to the SENIEUR protocol, it was found that the PHA-stimulated activation of AP-1 was commonly impaired in the elderly. In many of these, addition of phorbol ester partially compensated for this defect, but a minority remained refractory. The defect appeared to be in the amount of AP-1 activity produced, since the AP-1 protein that was produced by cells from old donors behaved in the same way as that from young donors and also contained c-fos and c-jun (103). Thereafter, the same group reported that both AP-1 and NF-AT were reduced in elderly donors´ stimulated T cells (104). However, whether these changes were associated with alterations in T cell subset composition was not reported. These data are consistent with those of Song et al . (92) demonstrating decreased c-jun mRNA but normal c-fos mRNA responses to PHA in T cells from elderly donors. Moreover, fewer lymphocytes from elderly donors exposed to influenza virus in vitro expressed fos and jun compared to cells of younger donors, possibly as a reflection of compromized activation of anti-viral responses (105). Amongst the transcription factors of known importance for IL 2 production, CD3-stimulated induction of NF-kappa B was also found to be decreased in old mice (106) and humans (107). One reason for insufficient NF-kB activation may be that the natural inhibitor I-kB is not adequately degraded because of compromized proteosome function [Ponnappan, cited in ref. (1)]. Age-associated inactivation of proteosome function has been independently reported and attributed to the effects oxidative damage, which can be partly prevented by hsp90 (108). Hsp 90 levels are themselves decreased with age, in T cells (109). Whisler et al . (104) also found reduced NF-kappa B in some elderly human donors´ stimulated T cells, but they did not find a correlation with depressed IL 2 production (unlike their findings with NF-AT, see above). Interpretations may be complicated, however, by the unexpected finding that NF-AT may exert negative regulatory, not stimulatory, effects on the immune response (110). Finally, in rats, Pahlavani et al . reported that the induction of AP-1, NF-kappa B and Oct-1 DNA binding activity in nuclear extracts of spleen cells from old animals was significantly lower than that of young animals, and the decrease of AP-1was due to reduction of c-fos mRNA, whereas c-jun remained the same in young and old cells (111). 5.3 Defects in costimulatory pathways Despite indications for the involvement of accessory cells in dysregulated immune function in the aged, most attention has been focussed on the T cell. T cells require stimulation via the antigen-specific TCR for activation. However, in addition they also require stimulation via non-polymorphic antigen-nonspecific costimulatory receptors. Abberations in these receptors would also lead to compromized T cell responses. Dobber et al . (112) reported that aged mouse CD4⁺ cells stimulated with Con A or anti-CD3 + anti-CD28 mAb did show decreased IL 2 production compared with young cells, but when stimulated with immobilized CD3 mAb alone, they produced more IL 2 than young cells (but still very little compared to CD3 + CD28). This suggests that aged CD4⁺ cells show diminished responsiveness to CD28 costimulation, whereas the limited response to stimulation via the TCR alone is retained in the old cells. Since memory cells in young (human) donors are easier to costimulate via CD28 than naive cells, whereas old donors´ cells are harder to stimulate with CD28 (113) this is consistent with alterations in aging not being solely explicable by accumulation of memory cells at the expense of naive cells. Moreover, in mice, the memory cells themselves function less well in old than in young donors (114-116). In this case, this may be related to their defective responses to signalling via the major costimulus receptor CD28, despite equivalent expression of CD28 on cells from young and old mice (117). Moreover, these cells, like certain other anergic (young) cells (118) may be able actively to suppress other cells in a mixed population, cells which otherwise would be capable of proliferation (119). Engwerda et al . have also more recently shown that activation-induced cell death (AICD) is increased in T cells from old mice, as a direct consequence of their decreased levels of CD28-mediated costimulation, which otherwise may protect stimulated cells from apoptosis (120) (although there are also other mechanisms of protection against apoptosis, some of which may not involve CD28 (121)) and conversely under certain circumstances CD28 costimulation may actually enhance apoptosis by upregulating apoptotic mediators such as bad (122). CD28 is a costimulator receptor twinned with a closely related second receptor CTLA-4 (CD152). This molecule delivers "off" signals to the T cell when ligated by the same structures as CD28 (CD80, CD86) (123). Old T cells may express increased amounts of CTLA-4, which may therefore make them harder to turn on even if CD28 functions normally (124). This is another property shared with young anergic T cells (A. Merl, unpublished results). Not only CD28 costimulatory mechanisms but other important accessory/adhesion pathways may be compromized in aging. Thus, Jackola et al . (125) reported defects in cell-cell binding amongst healthy elderly donors, which was associated with altered activation capacity of the integrin LFA-1. Moreover, other surface receptors implicated in costimulation may be downregulated with aging. Preliminary evidence is beginning to show that the density of expression of the CD40 ligand CD154 is decreased on T cells aged in vivo (Lio et al . Mech. Aging Dev., in press) and in vitro (M. Adibzadeh, unpublished results). 5.4 Alterations in cytokine production and response Once stimulated, T cells must transcribe T cell growth factor (TCGF) genes, secrete growth factors, upregulate TCGF receptors and respond to the cytokines. In this way, autocrine and/or paracrine clonal expansion, a prerequisite for successful immune responses, is effected. One result of poorer T cell function is decreased cytokine production. It has been long believed that a major dysfunction in T cells from elderly donors is a selectively decreased ability to secrete T cell growth factors. Many studies have confirmed that T cells from aged humans can also show defects in IL 2R expression, IL 2 secretion and DNA synthesis after stimulation with mitogens like PHA. Data on cytokine secretion in the earliest studies were controversial, perhaps mostly due to employing apparently healthy elderly donors without rigorously excluding underlying illness, nutritional or psychological status. In addition, cytokine secretion may be affected by other (non-pathological) parameters such as exercise, as reported by Shinkai et al . (126). They found that although the numbers of circulating CD3, CD4 and CD8 cells were similar in sedentary and exercising elderly donors, and proliferative responses and cytokine secretion were reduced, these parameters were significantly more compromized in the sedentary group. It was therefore anticipated that when data became available using donors selected according to standard criteria (namely, the SENIEUR protocol (127)), results on cytokine secretion and other immunogerontological parameters would become clearer. Such studies have confirmed dysregulation of cytokine production but not necessarily associated with lower IL 2 production. Thus, Sindermann et al . reported unchanged IL 2 and IFN-g production in elderly German SENIEUR donors, but they did find significantly decreased IFN-a and soluble IL 2 receptor secretion (128). Others have found that IFN-g production tended to be enhanced (18), and, as also shown in mouse, IL 10 production was enhanced as well (129,130). In vivo studies of plasma levels of factors such as IL 6 also reveal age-associated increases; in fact, it has been proposed that IL 6 levels may be a good overall biomarker of health in aging because plasma levels are correlated with functional status (131). Moreover, treating aged mice with IL 6- but not IL 1-neutralizing antibody resulted in a reversion of their cytokine production pattern to that characteristic of young animals (132). In American donors, Jackola et al . reported that the frequency of T cells responding to PHA by secreting IL 2 decreased with age, even in SENIEUR-selected donors (133). Clearly, despite the use of SENIEUR donors, discrepancies still arise. Unsuspected population genetic influences may be playing a role, since the distribution of MHC alleles differs even within different groups of the European population, and levels of immune responses and cytokine secretion as well as possibly longevity are known to be associated with MHC type (134). Nijhuis et al . agreed that IL 2 production in old Dutch SENIEUR donors (compared to young donors also selected with the SENIEUR protocol) was not decreased and presented evidence for increased IL 4 production. They also found that increased IL 4 production in elderly donors did not correlate simply with the measured increase in the fraction of memory (CD27-negative CD45RO⁺) cells, although this was confirmed to be the case for young donors (135). This suggests that in young donors, different levels of IL 4 production are determined solely by antigen exposure and amount of memory cells, but that in aged donors, other regulatory mechanisms are operating. In contrast, Candore et al . reported decreased IL 2 and IFN-g production but unaltered IL 4 and IL 6 secretion after PHA stimulation in Sicilian donors (136), and others have reported that both IL 2 and IL 4 secretion may be reduced even in (Italian) SENIEUR donors and have pointed out that decreased function may be linked to psychological factors (137), and suggested that these need to taken more into account in studies of immunosenescence (138). However, in this population, despite the relative decrease in cytokine secretion, T cells from these donors could proliferate well if supplied with exogenous growth factors (139). A more recent study with patients with Major Depression did not support a role for this condition in decreasing PHA-stimulated proliferative responses (140). Psychosocial factors may play a significant role in clinically-relevant situations also, eg. the humoral response to influenza vaccination. In one study, the effect of chronic stress (caring for a demented spouse) resulted in significantly lower antibody titers, as well as IL 1 and IL 2 production in the elderly caregivers (141). Some data are beginning to emerge on the regulatory effects of neuropeptides on cytokine secretion in young and elderly donors (142). Another study investigated the expression of dopamine receptors on human lymphocytes and revealed that precipitous loss of dopamine receptor D3 occurs between 40 - 50 years of age (143). Conversely, increasing levels of expression of cellular amyloid precursor protein (APP) by human lymphocytes are significantly positively associated with increasing age (144). Although the significance of these findings is unclear, studies of this type may begin to help shed some light on the mechanisms responsible for neuroimmunological communication and age-associated alterations. Other factors perhaps not sufficiently taken into account in previous studies may be not only differences between sexes but differences between females dependent on their reproductive history (145). Few studies have compared the behavior of T cells from young and old donors at the clonal level. Paganelli et al . (146) reported on T cell clones (TCC) obtained from two centenarians (a highly selected population) compared to those obtained from three young donors. CD4⁺ TCC made up 38% of TCC obtained from the young, but 53% of those from the old. Cytokine production from the CD8-TCC was the same in young and old-derived clones, but the CD4-TCC were different. Most TCC derived from young donors produced IFN-g but not IL 4, whereas those from the centenarians produced both. This was interpreted to indicate a shift in the CD4 population from predominantly Th1 to Th0 phenotype in the centenarians. Similarly, Kurashima et al . (147) found that, as expected, naive cells produced mainly IL 2 and memory cells mainly IL 4, in young mice. However, the reciprocal was observed in old mice: naive cells produced more IL 4 than memory cells and memory cells produced more IL 2 than naive cells, although overall levels were reduced in old compared to young mice. Thus, age-associated alerations in cytokine production are not determined solely by the subset changes, but by alterations within each of those subsets. On the other hand, although different investigators have confirmed the decrease in IL 2 production in old mice, they have concluded that this is solely a result of different subset composition. Thus, Engwerda et al . (148) reported that purified CD4 or CD8 cells from aged mice, stimulated with CD3-epsilon mAb and CD28 mAb, produced the same amounts of IL 2 and IL 4 as young cells of the same CD44^hi or CD44^lo phenotype (although they produced increased amounts of IFN-g ). Kirman et al . (149) reported that the age-associated increase in IL 4 secretion by mouse spleen cells was not caused by an increase in the numbers of IL 4-secreting cells, but the decrease of IL 2 secretion was associated with a decreased number of secretor cells. However, this could be prevented by exposing the mice constantly to high levels of IL 2 in vivo, which could therefore correct the age-associated cytokine imbalance in these mice. Decreased soluble IL 2R secretion has been noted in elderly donors and may be of greater significance than possible IL 2 secretion defects (150). Other defects in IL 2R expression in aged T cells have also been reported: they are defective in their upregulation of the high affinity receptor for IL 2 (151), and even that lower proportion of cells expressing the receptor and retaining their ability to internalise the IL 2 still fail to respond properly (152). Thus, even under conditions where IL 2 secretion is apparently normal, and where IL 2R expression is also apparently normal in terms of receptor affinity and number (153), aged T cells may still proliferate less vigorously than young cells, even in the presence of exogenous IL 2 (153). The reasons for such suboptimal proliferation are still not clear, and signal transduction defects by the cytokine receptors seem not to have been investigated intensively yet. Such intrinsic decreased proliferative capacity is even seen in T cell clones established from aged SENIEUR donors´ cells, particularly for CD8+ cells (154). On the other hand, the IL 2 secretion defect seen in vitro in many but not all studies may in fact be transient, with T cells from old donors re-acquiring this ability after a period in culture (155). Different donor states might then explain discrepancies found in cytokine secretion patterns even amongst SENIEUR donors, as noted above. Huang et al. found that old donors with apparent IL 2 secretion defects in vitro, in fact had relatively high serum IL 2 concentrations in vivo. Moreover, vaccination of young donors mimicked this effect and resulted in their T cells becoming refractory for IL 2 production shortly thereafter in vitro. These investigators therefore suggested that apparent defects in IL 2 secretion in elderly donors are a result of in vivo activation of their T cells by unknown mechanisms and reflect a normal event also seen in young donors after in vivo T cell activation by immunization (155). If these results are confirmed (and as far as the authors are aware, five years after publication this is not the case), a reassessment of the meaning of depressed IL 2 secretion by old T cells in vitro will be required. The immune "defect" observed here will then actually represent a normal consequence of activation, possibly a kind of refractory, temporary anergic state, or "exhaustion", which in animal models can even result in extra-thymic clonal deletion of the activated cells (156). On the other hand, "memory" cells accumulate in elderly donors, and may be in active division required for their maintenance of memory (157,158). Not only might these activated cells explain the data of Huang et al (155), but since they might eventually arrive at a post-mitotic state, this would also explain their eventual loss from the system altogether. Direct measurements of IL 2 levels in the serum of SENIEUR donors have also failed to detect age-associated decreases in one study (159), although a second study suggested that serum IL 2 levels were reduced in the very old (160). However, both studies agreed that the level of soluble IL 2R in the blood was increased in the elderly, which could contribute to decrease of IL 2 function. Other cytokines are also beginning to be examined, eg. spontaneous production of IL 8 by monocytes in vitro was reported to be lower in the elderly than in the young, but on stimulation with LPS more IL 8 was produced in elderly males than in young (161). Similarly, production of cytokines such as TNF-a may increase rather than decrease with age (162). Increases in TNF-a may be directly relevant for decreased T cell responses, because TNF-a can inhibit proliferation of some human TCC (163) and can attenuate TCR-signalling in vivo in mice (164). As well as altered cytokine levels in aging, altered levels of cytokine antagonists might also influence cytokine networks. These possibilities are now beginning to be explored (for factors in addition to sIL 2R mentioned above). Thus, Catania et al . (165) reported a study of 122 healthy aged compared to 39 unhealthy (urinary tract infections) and 100 young controls regarding plasma levels of IL 1R-antagonist and sTNF-R. These were higher in the healthy than in young controls, and were even higher in the infected subjects.

[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

5. MECHANISMS CONTRIBUTING TO IMMUNOSENESCENCE

5.1 Accessory cells

Decreased T cell responses in the elderly may be due to decreased T cell function, decreased accessory cell function, or both. There is evidence for accessory cell changes (73) and monocytes are clearly compromized in their function in the elderly, eg. in that they secrete less IL 1 and have decreased cytotoxicity and protein kinase translocation (74). A more recent analysis suggested that lipopolysaccharide (LPS)-stimulated monocytes from the elderly produced less G-CSF, GM-CSF, IL 8, TNF-a , and MIP-1-a as well as less IL 1ß compared to those from young donors (75). In some clinically-relevant animal models, it is the accessory cells which seem to contribute critically to age-associated suboptimal responses, eg. in the response of mice to trypanosome antigens (76). Another example where T and B cell function appears to be normal, but accessory cell function is compromized in aged mice comes from a vaccination model using pneumococcal preparations (77). To distinguish between T cell and APC alterations may be difficult but can be approached in certain models by using purified T cells in the absence of accessory cells. For example, using mitogenic CD2 mAb and soluble costimulatory factors (cytokines, phorbol esthers, mAb), Beckman et al . (78) have shown that in CD45RO⁺ CD4 cells, the only pathway not comparable between young and old donors was for stimulation by CD2 in combination with IL 7. Thus, signalling may be intact in old memory cells, except for IL 7-dependent pathways. In contrast, CD45RA⁺ cells from old donors responded less well than young naive cells to CD2 + IL 2, IL 6, IL 7, IL 1 or phorbol esther, suggesting multiple deficiencies in the naive cells but not the memory cells of old donors.

On the other hand, dendritic cells (DC) obtained from elderly persons are reported to be able to present antigen at least as well, if not better, than DC from young donors (79). The same group also reported that DC from the elderly were able to inhibit apoptosis and stimulate proliferation in pre-senescent cultured T cells (80). These results suggest that at least a subset of APC in the elderly retain optimal function. On the other hand, it must be borne in mind that these results were obtained using DC generated in vitro using IL 4 and GM-CSF. Since the production of GM-CSF in the elderly is decreased (see section 4.1) there may not be so many functional DC available in old donors.

5.2 Alterations in signal transduction

That the earliest events in T cell activation are compromized in the elderly is reflected in findings that cell surface alterations associated with activation are affected, eg. CD69 and CD71 upregulation does not take place (81). Incomplete T cell activation may be caused in the first instance by disturbed signal transduction. Particularly in T cells there are multiple levels where lesions might be sought, since signal transduction through one, several or all of the TCR, costimulatory receptors, or growth factor receptors could be compromized with age. There is evidence for alterations in all three of these possible loci.

Damage to the cytoskeleton paralleling aging may have profound effects on cell function. In the case of T cells this may be relevant even to the earliest stages of T cell activation, because, for example, the signal-transducing TCR zeta chain component is associated at the cell surface with the cytoskeleton (82). If this has something to do with receptor recycling or maintaining correct three-dimensional structure of the receptor, cytoskeletal dysfunction could have profound effects on TCR signal transduction. Certainly, initial biochemical events following TCR triggering (formation of second messengers such as IP3 and DAG) are compromized, although the activity of PLC (which is responsible for IP3 and DAG generation) appears conserved in old T cells (83). However, the actual amount of PLC present in freshly isolated cells may be decreased with aging (84). Antibody against the signal-transducing CD3 zeta chain precipitates a series of tyrosine-phosphorylated proteins, the levels of which decline with age (85). Therefore, the level of expression of the TCR components and/or their ability to transduce signals may be compromized in old T cells.

Signalling pathways mediated by the family of mitogen-activated protein kinases (MAPK) are considered essential for normal cell growth and function; CD3 stimulated human T cells from 50% of old subjects were found to show reductions in MAPK activation. Stimulation with phorbol esther in combination with calcium ionophore resulted in greater MAPK activation in old cells, but still not to the same extent as young cells (86), suggesting signalling deficits between the TCR and the inducers of MAPK. MAPK are activated by another protein kinase, MEK; in mouse CD4 cells, there is an age-associated decrease in MEK (87). Moreover, kinases are commonly counter-regulated by phosphatases, and even if kinase decrease were not to occur, increase in phosphatase activity might have the same result. In the case of MAPK, it has been reported that expression of MAP phosphatase is indeed increased (in aged rat hepatocytes, at least) (88). In T cells, signalling through the TCR, CD4, CD8 or the IL 2R resulted in lowered protein tyrosine kinase activity in cells from old compared to young donors, although direct activation of protein tyrosine kinases (PTK) by pervanadate was normal in the old (89). It is therefore not yet clear whether the age-related decreased tyrosine phosphorlylation observed in CD3-stimulated human T cells is related to changes in PTKs or phosphatases (PTPases). Recent data from Whisler et al . indicate that CD45-PTPase activity in old cells after CD3-stimulation is not increased compared to young cells (90). They further found that fyn enzymatic activity but not lck activity was reduced in a high proportion of T cells from the elderly compared to the young, although protein levels were the same. They concluded that decreased fyn activation but not increased PTPase activity may contribute to lowered responses in the elderly (90).

Cell cycle analyses of PHA-stimulated cells from aged donors indicate a decreased frequency of cells entering S-phase with this age-related impairment of G1 progression correlating with decreased expression of c-jun, c-myc, c-myb, IL 2 and CD25 (91-93). The proportion of cells expressing c-myc (G0 to G1 marker) and c-myb (G1 to S marker) was decreased after PHA stimulation of old T cells, but the amount per cell seemed to remain the same as in young T cells (93). T cells retaining antigen recognition and effector function, yet apparently in a post-mitotic senescent or pre-senescent state have been described (94). These investigators also demonstrated that aged human T cells paralleled the senescent phenotype of fibroblasts in that on restimulation, fewer cells responded by entering the cell cycle, the remainder being arrested before S-phase. The cell cycle was also prolonged in those ca. 20% of senescent cells which could be restimulated (95). At least some of these results may reflect the situation in vivo, where PHA stimulation resulted in an earlier accumulation of cells in S phase in young donors´ T cells, and a significant delay, but eventually equivalent level, of S phase cells in the elderly (96).

In Fischer rats, the age-associated decrease in IL 2 mRNA and protein correlates with a decreasing ability of nuclear extracts of freshly isolated T cells to bind an oligonucleotide representing the transcription factor NF-AT (97), suggesting differences in transcriptional regulation in young and old cells. NF-AT forms an important family of at least four transcription factors; NF-AT DNA binding activity has been found in nuclear extracts of stimulated T cells (98) and is thought to be important for IL 2 gene transcription (99).

A set of transcription factors involving complexes of the various c-jun and c-fos proteins is involved in regulating transcription of many genes, including IL 2, and activation of AP-1 is detected a few hours after T cell stimulation (100,101). Specific defects in AP-1 activation have been reported in young T cell clones rendered anergic in vitro (102). The anergic phenotype is in some ways similar to the senescent phenotype (ie. cells can be stimulated via the TCR to secrete cytokines, be cytotoxic, but they cannot expand clonally via autocrine IL 2 production). AP-1 activation may be impaired in in vivo-aged human T cells as well (103). Using T cells from elderly donors rigorously selected for good health according to the SENIEUR protocol, it was found that the PHA-stimulated activation of AP-1 was commonly impaired in the elderly. In many of these, addition of phorbol ester partially compensated for this defect, but a minority remained refractory. The defect appeared to be in the amount of AP-1 activity produced, since the AP-1 protein that was produced by cells from old donors behaved in the same way as that from young donors and also contained c-fos and c-jun (103). Thereafter, the same group reported that both AP-1 and NF-AT were reduced in elderly donors´ stimulated T cells (104). However, whether these changes were associated with alterations in T cell subset composition was not reported. These data are consistent with those of Song et al . (92) demonstrating decreased c-jun mRNA but normal c-fos mRNA responses to PHA in T cells from elderly donors. Moreover, fewer lymphocytes from elderly donors exposed to influenza virus in vitro expressed fos and jun compared to cells of younger donors, possibly as a reflection of compromized activation of anti-viral responses (105). Amongst the transcription factors of known importance for IL 2 production, CD3-stimulated induction of NF-kappa B was also found to be decreased in old mice (106) and humans (107). One reason for insufficient NF-kB activation may be that the natural inhibitor I-kB is not adequately degraded because of compromized proteosome function [Ponnappan, cited in ref. (1)]. Age-associated inactivation of proteosome function has been independently reported and attributed to the effects oxidative damage, which can be partly prevented by hsp90 (108). Hsp 90 levels are themselves decreased with age, in T cells (109). Whisler et al . (104) also found reduced NF-kappa B in some elderly human donors´ stimulated T cells, but they did not find a correlation with depressed IL 2 production (unlike their findings with NF-AT, see above). Interpretations may be complicated, however, by the unexpected finding that NF-AT may exert negative regulatory, not stimulatory, effects on the immune response (110). Finally, in rats, Pahlavani et al . reported that the induction of AP-1, NF-kappa B and Oct-1 DNA binding activity in nuclear extracts of spleen cells from old animals was significantly lower than that of young animals, and the decrease of AP-1was due to reduction of c-fos mRNA, whereas c-jun remained the same in young and old cells (111).

5.3 Defects in costimulatory pathways

Despite indications for the involvement of accessory cells in dysregulated immune function in the aged, most attention has been focussed on the T cell. T cells require stimulation via the antigen-specific TCR for activation. However, in addition they also require stimulation via non-polymorphic antigen-nonspecific costimulatory receptors. Abberations in these receptors would also lead to compromized T cell responses. Dobber et al . (112) reported that aged mouse CD4⁺ cells stimulated with Con A or anti-CD3 + anti-CD28 mAb did show decreased IL 2 production compared with young cells, but when stimulated with immobilized CD3 mAb alone, they produced more IL 2 than young cells (but still very little compared to CD3 + CD28). This suggests that aged CD4⁺ cells show diminished responsiveness to CD28 costimulation, whereas the limited response to stimulation via the TCR alone is retained in the old cells. Since memory cells in young (human) donors are easier to costimulate via CD28 than naive cells, whereas old donors´ cells are harder to stimulate with CD28 (113) this is consistent with alterations in aging not being solely explicable by accumulation of memory cells at the expense of naive cells. Moreover, in mice, the memory cells themselves function less well in old than in young donors (114-116). In this case, this may be related to their defective responses to signalling via the major costimulus receptor CD28, despite equivalent expression of CD28 on cells from young and old mice (117). Moreover, these cells, like certain other anergic (young) cells (118) may be able actively to suppress other cells in a mixed population, cells which otherwise would be capable of proliferation (119). Engwerda et al . have also more recently shown that activation-induced cell death (AICD) is increased in T cells from old mice, as a direct consequence of their decreased levels of CD28-mediated costimulation, which otherwise may protect stimulated cells from apoptosis (120) (although there are also other mechanisms of protection against apoptosis, some of which may not involve CD28 (121)) and conversely under certain circumstances CD28 costimulation may actually enhance apoptosis by upregulating apoptotic mediators such as bad (122). CD28 is a costimulator receptor twinned with a closely related second receptor CTLA-4 (CD152). This molecule delivers "off" signals to the T cell when ligated by the same structures as CD28 (CD80, CD86) (123). Old T cells may express increased amounts of CTLA-4, which may therefore make them harder to turn on even if CD28 functions normally (124). This is another property shared with young anergic T cells (A. Merl, unpublished results).

Not only CD28 costimulatory mechanisms but other important accessory/adhesion pathways may be compromized in aging. Thus, Jackola et al . (125) reported defects in cell-cell binding amongst healthy elderly donors, which was associated with altered activation capacity of the integrin LFA-1. Moreover, other surface receptors implicated in costimulation may be downregulated with aging. Preliminary evidence is beginning to show that the density of expression of the CD40 ligand CD154 is decreased on T cells aged in vivo (Lio et al . Mech. Aging Dev., in press) and in vitro (M. Adibzadeh, unpublished results).

5.4 Alterations in cytokine production and response

Once stimulated, T cells must transcribe T cell growth factor (TCGF) genes, secrete growth factors, upregulate TCGF receptors and respond to the cytokines. In this way, autocrine and/or paracrine clonal expansion, a prerequisite for successful immune responses, is effected. One result of poorer T cell function is decreased cytokine production. It has been long believed that a major dysfunction in T cells from elderly donors is a selectively decreased ability to secrete T cell growth factors. Many studies have confirmed that T cells from aged humans can also show defects in IL 2R expression, IL 2 secretion and DNA synthesis after stimulation with mitogens like PHA. Data on cytokine secretion in the earliest studies were controversial, perhaps mostly due to employing apparently healthy elderly donors without rigorously excluding underlying illness, nutritional or psychological status. In addition, cytokine secretion may be affected by other (non-pathological) parameters such as exercise, as reported by Shinkai et al . (126). They found that although the numbers of circulating CD3, CD4 and CD8 cells were similar in sedentary and exercising elderly donors, and proliferative responses and cytokine secretion were reduced, these parameters were significantly more compromized in the sedentary group.

It was therefore anticipated that when data became available using donors selected according to standard criteria (namely, the SENIEUR protocol (127)), results on cytokine secretion and other immunogerontological parameters would become clearer. Such studies have confirmed dysregulation of cytokine production but not necessarily associated with lower IL 2 production. Thus, Sindermann et al . reported unchanged IL 2 and IFN-g production in elderly German SENIEUR donors, but they did find significantly decreased IFN-a and soluble IL 2 receptor secretion (128). Others have found that IFN-g production tended to be enhanced (18), and, as also shown in mouse, IL 10 production was enhanced as well (129,130). In vivo studies of plasma levels of factors such as IL 6 also reveal age-associated increases; in fact, it has been proposed that IL 6 levels may be a good overall biomarker of health in aging because plasma levels are correlated with functional status (131). Moreover, treating aged mice with IL 6- but not IL 1-neutralizing antibody resulted in a reversion of their cytokine production pattern to that characteristic of young animals (132). In American donors, Jackola et al . reported that the frequency of T cells responding to PHA by secreting IL 2 decreased with age, even in SENIEUR-selected donors (133). Clearly, despite the use of SENIEUR donors, discrepancies still arise. Unsuspected population genetic influences may be playing a role, since the distribution of MHC alleles differs even within different groups of the European population, and levels of immune responses and cytokine secretion as well as possibly longevity are known to be associated with MHC type (134). Nijhuis et al . agreed that IL 2 production in old Dutch SENIEUR donors (compared to young donors also selected with the SENIEUR protocol) was not decreased and presented evidence for increased IL 4 production. They also found that increased IL 4 production in elderly donors did not correlate simply with the measured increase in the fraction of memory (CD27-negative CD45RO⁺) cells, although this was confirmed to be the case for young donors (135). This suggests that in young donors, different levels of IL 4 production are determined solely by antigen exposure and amount of memory cells, but that in aged donors, other regulatory mechanisms are operating. In contrast, Candore et al . reported decreased IL 2 and IFN-g production but unaltered IL 4 and IL 6 secretion after PHA stimulation in Sicilian donors (136), and others have reported that both IL 2 and IL 4 secretion may be reduced even in (Italian) SENIEUR donors and have pointed out that decreased function may be linked to psychological factors (137), and suggested that these need to taken more into account in studies of immunosenescence (138). However, in this population, despite the relative decrease in cytokine secretion, T cells from these donors could proliferate well if supplied with exogenous growth factors (139). A more recent study with patients with Major Depression did not support a role for this condition in decreasing PHA-stimulated proliferative responses (140). Psychosocial factors may play a significant role in clinically-relevant situations also, eg. the humoral response to influenza vaccination. In one study, the effect of chronic stress (caring for a demented spouse) resulted in significantly lower antibody titers, as well as IL 1 and IL 2 production in the elderly caregivers (141). Some data are beginning to emerge on the regulatory effects of neuropeptides on cytokine secretion in young and elderly donors (142). Another study investigated the expression of dopamine receptors on human lymphocytes and revealed that precipitous loss of dopamine receptor D3 occurs between 40 - 50 years of age (143). Conversely, increasing levels of expression of cellular amyloid precursor protein (APP) by human lymphocytes are significantly positively associated with increasing age (144). Although the significance of these findings is unclear, studies of this type may begin to help shed some light on the mechanisms responsible for neuroimmunological communication and age-associated alterations. Other factors perhaps not sufficiently taken into account in previous studies may be not only differences between sexes but differences between females dependent on their reproductive history (145).

Few studies have compared the behavior of T cells from young and old donors at the clonal level. Paganelli et al . (146) reported on T cell clones (TCC) obtained from two centenarians (a highly selected population) compared to those obtained from three young donors. CD4⁺ TCC made up 38% of TCC obtained from the young, but 53% of those from the old. Cytokine production from the CD8-TCC was the same in young and old-derived clones, but the CD4-TCC were different. Most TCC derived from young donors produced IFN-g but not IL 4, whereas those from the centenarians produced both. This was interpreted to indicate a shift in the CD4 population from predominantly Th1 to Th0 phenotype in the centenarians. Similarly, Kurashima et al . (147) found that, as expected, naive cells produced mainly IL 2 and memory cells mainly IL 4, in young mice. However, the reciprocal was observed in old mice: naive cells produced more IL 4 than memory cells and memory cells produced more IL 2 than naive cells, although overall levels were reduced in old compared to young mice. Thus, age-associated alerations in cytokine production are not determined solely by the subset changes, but by alterations within each of those subsets. On the other hand, although different investigators have confirmed the decrease in IL 2 production in old mice, they have concluded that this is solely a result of different subset composition. Thus, Engwerda et al . (148) reported that purified CD4 or CD8 cells from aged mice, stimulated with CD3-epsilon mAb and CD28 mAb, produced the same amounts of IL 2 and IL 4 as young cells of the same CD44^hi or CD44^lo phenotype (although they produced increased amounts of IFN-g ). Kirman et al . (149) reported that the age-associated increase in IL 4 secretion by mouse spleen cells was not caused by an increase in the numbers of IL 4-secreting cells, but the decrease of IL 2 secretion was associated with a decreased number of secretor cells. However, this could be prevented by exposing the mice constantly to high levels of IL 2 in vivo, which could therefore correct the age-associated cytokine imbalance in these mice.

Decreased soluble IL 2R secretion has been noted in elderly donors and may be of greater significance than possible IL 2 secretion defects (150). Other defects in IL 2R expression in aged T cells have also been reported: they are defective in their upregulation of the high affinity receptor for IL 2 (151), and even that lower proportion of cells expressing the receptor and retaining their ability to internalise the IL 2 still fail to respond properly (152). Thus, even under conditions where IL 2 secretion is apparently normal, and where IL 2R expression is also apparently normal in terms of receptor affinity and number (153), aged T cells may still proliferate less vigorously than young cells, even in the presence of exogenous IL 2 (153). The reasons for such suboptimal proliferation are still not clear, and signal transduction defects by the cytokine receptors seem not to have been investigated intensively yet. Such intrinsic decreased proliferative capacity is even seen in T cell clones established from aged SENIEUR donors´ cells, particularly for CD8+ cells (154).

On the other hand, the IL 2 secretion defect seen in vitro in many but not all studies may in fact be transient, with T cells from old donors re-acquiring this ability after a period in culture (155). Different donor states might then explain discrepancies found in cytokine secretion patterns even amongst SENIEUR donors, as noted above. Huang et al. found that old donors with apparent IL 2 secretion defects in vitro, in fact had relatively high serum IL 2 concentrations in vivo. Moreover, vaccination of young donors mimicked this effect and resulted in their T cells becoming refractory for IL 2 production shortly thereafter in vitro. These investigators therefore suggested that apparent defects in IL 2 secretion in elderly donors are a result of in vivo activation of their T cells by unknown mechanisms and reflect a normal event also seen in young donors after in vivo T cell activation by immunization (155). If these results are confirmed (and as far as the authors are aware, five years after publication this is not the case), a reassessment of the meaning of depressed IL 2 secretion by old T cells in vitro will be required. The immune "defect" observed here will then actually represent a normal consequence of activation, possibly a kind of refractory, temporary anergic state, or "exhaustion", which in animal models can even result in extra-thymic clonal deletion of the activated cells (156). On the other hand, "memory" cells accumulate in elderly donors, and may be in active division required for their maintenance of memory (157,158). Not only might these activated cells explain the data of Huang et al (155), but since they might eventually arrive at a post-mitotic state, this would also explain their eventual loss from the system altogether.

Direct measurements of IL 2 levels in the serum of SENIEUR donors have also failed to detect age-associated decreases in one study (159), although a second study suggested that serum IL 2 levels were reduced in the very old (160). However, both studies agreed that the level of soluble IL 2R in the blood was increased in the elderly, which could contribute to decrease of IL 2 function.

Other cytokines are also beginning to be examined, eg. spontaneous production of IL 8 by monocytes in vitro was reported to be lower in the elderly than in the young, but on stimulation with LPS more IL 8 was produced in elderly males than in young (161). Similarly, production of cytokines such as TNF-a may increase rather than decrease with age (162). Increases in TNF-a may be directly relevant for decreased T cell responses, because TNF-a can inhibit proliferation of some human TCC (163) and can attenuate TCR-signalling in vivo in mice (164).

As well as altered cytokine levels in aging, altered levels of cytokine antagonists might also influence cytokine networks. These possibilities are now beginning to be explored (for factors in addition to sIL 2R mentioned above). Thus, Catania et al . (165) reported a study of 122 healthy aged compared to 39 unhealthy (urinary tract infections) and 100 young controls regarding plasma levels of IL 1R-antagonist and sTNF-R. These were higher in the healthy than in young controls, and were even higher in the infected subjects.