Age-related changes of signal transduction in T cells

Experimental Gerontology, Vol. 34, No. 1, pp. 7–18, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0531-5565/99 $–see front matter

PII S0531-5565(98)00067-9

REVIEW

AGE-RELATED CHANGES OF SIGNAL TRANSDUCTION IN T CELLS

KATSUIKU HIROKAWA Department of Pathology and Immunology, School of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku Tokyo, Japan

INTRODUCTION THE DEFENSE SYSTEM for combatting infectious agents in mammals is composed of the innate and the acquired immune systems. Macrophages, granulocytes, and natural killer (NK) cells are three major components in the innate immune system, while lymphocytes are one major component in the acquired immune system. Lymphocytes are composed of two major subgroups, T cells and B cells, and both of these are composed of an innumerable number of clones. Each clone is small in number shortly after the birth, but can quickly increase in number by exposure to microbes (infection, in other words) in the environment. This clonal expansion of lymphocytes is induced by antigenic stimulation from the outer environment, and is a very important process for efficient execution of immune function against infectious agents. In other words, receptors of lymphocytes recognizing antigenic epitopes play a major role in the development and operation of immune function. Through the receptors, antigenic stimulation can be transduced into intracellular signaling, eventually leading to cell proliferation or production of cytokines. This intracellular signal pathway is composed of chain reactions of multiple molecules, and any disorders of any molecules can give rise to impairment of T cell functions such as proliferation and cytokine production. In fact, such an alteration can occur during the course of aging. Aging of the immune system mainly occurs in T cells The innate immune system starts to immediately function after birth, and does not manifest a distinct age change throughout the life. In contrast, the acquired immune system, mainly composed of lymphocytes, is still immature at the time of birth and quickly develops by the

Correspondence to: K. Hirokawa, M.D., Department of Pathology and Immunology, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku Tokyo 113-8519, Japan. Tel: 181-3-5803-5173; Fax: 181-3-3813-1790; E-mail: [email protected] (Received 11 August 1998; Accepted 8 October 1998) 7

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exposure to innumerable antigens in the outer environment (Hirokawa, 1998). The immunological vigor quickly increases in the very early phase of the life, peaks at puberty, but starts to decline thereafter. Studies in many laboratories including ours (Makinodan and Kay, 1980; Wick and Grubeck-Loebenstein, 1997; Hirokawa, 1998) indicated that (1) age-related decline of immunological functions mainly occurs in T cells; (2) the thymic involution precedes the onset of decline of T cell functions; (3) activity of NK cells also declines with age, but less than that of T cells; and (4) alteration of B cells, macrophages, and granulocytes is marginal. T cell functions showing age changes are proliferation to mitogenic and allogeneic stimulation (Hirokawa et al., 1984), helper function in antibody response (Hirokawa et al., 1994), delayed type hypersensitivity response (Bender and Tallman, 1992), cytokine production (Thoman and Weigle, 1981; Hara et al., 1987), and cytotoxic function (Bender and Tallman, 1982; Hirokawa et al., 1994). It is interesting to note that all these changes of T cells are preceded by thymic involution. Although shrinkage of the thymus in size (thymic involution) starts to be observed at around puberty, the immunological thymic function to promote T cell differentiation begins to decline shortly after the birth (Hirokawa et al., 1994). Aging of T cells as reflected in number, subsets, and quality The age-related changes of T cells are seen in the following three contexts: (1) a decrease in T cell number; (2) a change in the composition of T cell subsets; and (3) qualitative changes such as proliferation and cytokine production (Hirokawa, 1998). 1) Number. In healthy people, the number of peripheral T cells shows a significant decline between the second and third decades. It stays at almost the same level through the sixth decade and declines after the seventh decade. The number of peripheral T cells is easily decreased by infection and stress, and the recovery to normal levels occurs in young, but not in old, individuals. Such retardation in the recovery is most likely due to age-related loss of thymic function mentioned above. 2) Subsets. The number of CD41 T cells stays relatively constant in healthy adult and senescent stages, while that of CD81 T cells gradually declines with age (Utsuyama et al., 1992). CD41 T cells can be divided into two subsets by surface markers; CD41CD45RA1 (naive T cells) and CD41CD45RO1 (or CD41CD291, memory T cells) (Ernst et al., 1990). During the aging process, individuals are exposed to various antigenic stimulation. Thus, continuous activation, clonal expansion, and elimination of T cells of various specificities eventually lead to changes in the T cell repertoire. One outstanding age-related change in T cell subsets is a decrease of naive T cells with a concomitant increase of memory T cells. The change is observed not only in the human but also in the mouse model (Utsuyama et al., 1992; GrubeckLoebenstein, 1997). It is interesting to note that a T cell receptor oligoclonality develops in the aged individuals. Most old mice and human beings contain large clones of CD81abTCR1 T cells (Ku et al., 1997; Schwab et al., 1997; Ricalton et al., 1998). After birth, percentages and absolute numbers of circulating CD72 T cells (CD41CD45RO1CD45RA2) increase significantly during aging of human. Expansion of CD72 T cells in vivo has been found in certain diseases associated with chronically repeated T cell stimulation (Reinhold and Abken, 1997). Another prominent age-related change of T cells subsets is an increase of extrathymic T cells as a compensation for decreased thymic T cells. The increase of extrathymic IL2Rb1NK12TCRintermediate cells in liver and periphery could be closely related to immunological

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changes with aging (Tsukahara et al., 1997). An oligoclonality is also observed in this case, as the CD8ab1TCRa(b)intermediate liver lymphocytes in aged nu/nu mice preferentially use TCRVb5 and/or TCRVb8 (Emoto et al., 1997). With aging, a unique CD41CD81 IEL may expand at a local site of the intestine under the influence of intestinal microflora, and may contribute to the first line of defense against various pathogens in the epithelium (Takimoto et al., 1992). 3) Qualitative changes. Two important age-related qualitative changes in T cells are decreased capacity for proliferation and alteration of cytokine production (Hirokawa, 1998). These changes directly reflect the age changes of T cell subsets as mentioned above, and are partly caused by repeated antigenic activation. However, changes in proliferation and cytokine production are also observed in T cells from TCR transgenic mice, although the specific antigen recognized by this transgenic T cells does not appear in the environment (Haynes et al., 1997). These findings support the presence of intrinsic qualitative change of old T cells that do not depend on response to antigen. One most possible intrinsic qualitative change in T cells during aging is the age-related change of intracellular signal transduction. Signal transduction related to T cell activation There are two major routes of stimulation causing proliferation and cytokine production in T cells. One is the T cell receptor (TCR), and the other is the cytokine receptor (Fig. 1). The TCR is a complex that is composed of eight molecules. Antigen is recognized by the ab (or gd) heterodimer, which needs an additional three kinds of accessory molecules for transmission of antigenic signals into the cytoplasm. These accessory molecules are the CD3 complex, composed of three kinds of dimers, ge, de, and zz (Saito, 1996). CD3 molecules have long cytoplasmic portions, possessing tyrosine activated motifs (TAM). Tyrosine residues within the motifs are phosphorylated after recognition of antigen by TCR, and then sequentially gives rise to the activation of tyrosine kinases associated with TCR complex and CD4/8 molecules such as lck, fyn, and ZAP-70. These tyrosine kinases are thought to play an important role in the signal transduction from TCR to various signal molecules in the downstream pathway. Various cytokines operate as growth factors or factors to induce protein synthesis. Among them, IL-2 is a well-known cytokine causing T cell proliferation. Two types of molecules play a major role in the signal transduction from cytokine receptors to the nucleus. They are JAK kinases (JAK-1, -2, -3 and Tyk-2), and signal transducers and activators of transcription (STAT) proteins (STAT-1, 2, 3, 4, 5 and 6). The type of JAK kinases and STAT proteins employed by receptors for different cytokines are different (Taniguchi, 1995). T cell activation does not occur following one signal from either the TCR or cytokine receptor, but needs an additional second signal from costimulation via other surface molecules. One of the important surface molecules in this regard is CD28. In any event, any alteration occurring in any of the molecules composing receptors or signaling molecules is likely to downregulate the intracellular signal transduction, eventually resulting in the impairment of T cell functions (Fig. 1). T cell receptor (TCR) Aside from some anticipated clonal expansions induced by antigenic stimulation, the ageassociated alterations in immune functions are not caused by any profound changes in the overall TCR repertoire (Gonzalez-Quintial et al., 1995).

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FIG. 1. Schematic presentation of signal pathways in T cells. Abbreviations: MHC, major histocompatibility complex; PIP, phosphatidyl inositol 4-phosphate; PIP2, phosphatidyl inositol 4-5 bisphosphate; IP3, inositol 1-4-5 trisphosphate; DAG, diacyl glycerol; PLCg1, phospholipase Cg1; ER, endoplasmic reticulum; PKC, protein kinase C; TCR, T cell receptor; CaM, calmodulin; CN, calcineurin; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; JNK, Jun N-terminal kinase; STAT, signal transducers and activators of transcription; PI3 kinase, phosphoinositide 3-kinase; protein tyrosine kinase, ZAP-70, fyn, lck. Asterisks indicate molecules showing age-related changes.

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The phosphorylation of the CD3-z chain of CD41T cells from aged mice exhibits abnormalities (decreased phosphorylation), both in the resting state and after the activation process (Garcia and Miller, 1997), although the phosphorylation of CD3 z-associated ZAP-70 changes only slightly after stimulation of T cells (Miller et al., 1997). However, Utsuyama et al., (1997) reported that the phosphorylation of ZAP-70 and fyn after activation significantly decreased in T cells from old mice compared with T cells from young mice. A marked decrease in the expression of ZAP-70 was also observed in a study using T cell clone established from old mice (Zeng et al., 1996). Quadri et al., (1996) reported that the intrinsic activity of the enzymes is preserved, and that the age-associated defect in PTK activation occurs as a consequence of an upstream biochemical alteration. Expression of the z chain of the TCR complex decreased or disappeared after oxidative stress (Otsuji et al., 1996), and the same thing is likely to happen during aging. Cytokines and their receptors An alteration of composition of T cell subsets appears to be related to a change in cytokine production. Production of IL-2 is known to decrease with age (Thoman and Weigle, 1981; Hirokawa et al., 1984), while other cytokines such as IL-6 are reported to increase with age (Hara et al., 1987; Paganelli et al., 1994; Roubenoff et al., 1998)). Impairment of cytokine receptors may be responsible for dysfunction of old T cells, as no improvement in the proliferative response of T cells from aged mice is found following the addition of any cytokine (IL-2, IL-4, IFNg, IL-1a, or IL6) (Pahlavani et al., 1998). IL-2 receptor-bearing splenic T lymphocytes derived from aged C57BL6/J mice (22–24 months) display a relative inability to respond to IL-2 when compared to similar cells from young (2–3 months) animals (Thoman, 1991). A subpopulation of T lymphocytes from aged mice did not express a sufficient density of high affinity IL-2R as a consequence of mitogenic activation (Proust et al., 1988). The soluble form of the cytokine receptor is known to increase in the elderly; for example, elevated serum sIL-6R is one of the characteristics of aged MRL/lpr mice, and can mediate the IL-6 functions through the IL-6 signal-transducing receptor component gp130, contributing to development of autoimmune disease in MRL/lpr mice (Suzuki et al., 1993). Weber et al., (1997) reported that the age-dependent decline in T cell functions is due to an age-related defect in signal transduction, because functional expression of receptors displayed by aged T-cells is not reduced. This opinion may be right in T cells in the resting stage. In T cells after activation, however, we found that reexpression of receptors (TCR and IL-2R) is lower and much more retarded in aged T cells compared with young T cells (Wakikawa et al., 1997). CD28 CD28 on T cells mediates an essential costimulatory signal following stimulation of the T cell receptor (TCR). An increase in the proportion of CD282 T cells is observed during aging and in situations of chronic immune stimulation (Effros, 1997; Merino et al., 1998). T cells showing replicative senescence in culture no longer express CD28, suggesting that CD282 T cells observed in vivo may be the progeny of memory cells that have been repeatedly stimulated (Effros, 1997). After mitogenic stimulation, percentage of CD281 T cells increases regardless of age, but the rate of increase is significantly lower in old than in young T cells. In addition,

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the amount of CD28 expressed is also lower in old than in young T cells after activation (Wakikawa et al., 1997). Anti-CD28 –mediated costimulation was found to rescue T cells from young mice from anti-CD3e–induced cell death, but did not rescue T cells from aged mice. This failure of T cells from aged mice to respond to costimulatory signals appears to contribute to the decreased proliferation observed from cultures containing these cells (Engwerda et al., 1996). Calcium defect Intracellular free Ca21 influx is an important step for T cell activation. Many investigators reported that T cells from old mice exhibit lower average rises in calcium concentration than T cells from young donors when stimulated with either mitogenic lectins or anti-CD3e (Utsuyama et al., 1993, 1997; Miller, 1996; Zeng et al., 1996). Among T cell subsets, Pgp-11 T cells (appearing to be memory T cells) from both young and old mice generate poor Ca21 responses (Philosophe and Miller, 1990). However, Naylor et al., (1992) reported that no abnormality of intracellular free Ca21 fluxes could be detected in purified T cells from elderly subjects when stimulated with the anti-CD3 antibody. Nevertheless, the proliferative responses of purified T cells to phorbol ester (PMA) and calcium ionophore (INM) was defective in the elderly subjects. This report is inconsistent with many others, because most articles reported that the combined use of PMA and INM could restore the decreased response of T cells from aged subjects. Impairment of early signaling pathways is responsible Because PMA can directly activate PKC, and INM can induce calcium influx, the combination of PMA and INM can bypass the early signaling pathway of T cell activation. Accordingly, the proliferative response of T cells following the combined use of PMA and INM becomes comparable between young and aged mice, while the Con A response remained depressed in aged mice. These results suggest that the impairment of early (but not late) events may be responsible for the suppression of T cell proliferation in aged animals (Saini and Sai, 1993; Quadri et al., 1996; Utsuyama et al., 1997). In our study, T cell clones established from old mice showed impaired proliferation after stimulation with anti-CD3 antibody, but were fully activated to the level of a T cell clone from a young mouse by stimulation with PMA plus INM. However, splenic T cells freshly prepared from old mice did not show full recovery after the same treatment. The results indicate that one major blockade in the signal transduction of T cells from old mice is present in the pathway just after TCR, but besides this, the blockade is also present in multiple downstream sites (Fig. 1), which cannot be bypassed by stimulation with PMA plus INM (Quadri et al., 1996; Utsuyama et al., 1997). Beckman et al., (1995) also suggested the presence of multiple signaling deficiencies in the proliferation of human CD4 T cells after stimulation with the anti-CD2 antibody. Second messengers and phospholipase Inositol trisphosphate (IP3) plays a major role in the intracellular free Ca21 influx. IP3, together with diacylglycerol (DAG), is generated from phosphatidylinositol 4,5-bisphosphate (PIP2) by the enzymatic activity of phospholipaseg1 (PLCg1). The formation of IP3 is decreased in T cells from old mice compared with those from young mice (Utsuyama et al., 1993; Kawasnishi, 1993). DAG is responsible for activation of PKC, and is known to also decrease with age (Utsuyama et al., 1993). PLCg1 play an important role in the liberation of second messengers in T cells (Secrist et al.,

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1991). When PLCg1 was extracted from T cells, no difference was detected in the activity between young and old mice (Utsuyama et al., 1993). However, tyrosine phosphorylation of PLCg1 after activation was decreased in T-lymphocytes from old mice (Grossmann et al., 1995; Utsuyama et al., 1997). So some molecules that are present upstream of PLCg1 become defective during aging, as described later. Some of the most likely molecules would be protein tyrosine kinases (PTK) associated with TCR. Protein tyrosine kinases (PTK) Optimal signal transduction through the TCR/CD3 complex requires the coordinated activities of protein tyrosine kinases (PTKs) fyn and lck in addition to protein tyrosine phosphatases (PTPases) such as CD45 (Whisler et al., 1997). Many investigators reported that the enzymatic activity of PTKs was lower in T cells from old than in T cells from young subjects. However, the types of PTKs showing impairment were different according to different investigators; i.e., lck and ZAP-70 (Liu et al., 1997), fyn (Whisler et al., 1997), fyn and ZAP-70 (Utsuyama et al., 1997; Pahlavani et al., 1998), and ZAP-70 (Chakravarti et al., 1998). It is of interest that the decline in activities of these signaling molecules with age was not associated with changes in their corresponding protein levels (Liu et al., 1997; Utsuyama et al., 1997). Although signaling defects have been recognized in PTKs, their biochemical nature is still not fully understood. The increased sensitivity of cells from senescent organisms to PTK inhibitors is most likely related to a lesser PTK activity, because a significant decrease in the tyrosine phosphorylation of particular endogenous substrates was observed as a consequence of either CD3, CD4, CD8, or IL-2R activation (Quadri et al., 1996). However, no age-related difference in tyrosine phosphorylation could be demonstrated when T cells were activated by pervanadate, a pharmacological activator of PTK. These results suggest that the intrinsic activity of the enzymes is preserved, and that the age-associated defect in PTK activation occurs as a consequence of an upstream biochemical alteration. One of possible site is the CD3z chain, as described in the section on TCR. Protein kinase C (PKC) PKC is a key molecule in intracellular signal transduction. PKCa, b d, e, and z isoforms are all present in human T cells from young or elderly subjects. The translocation of PKC-a, -b, -d, and -e can be observed under anti-CD3 mAb stimulation in lymphocytes of young subjects, while in the case of elderly subjects only the PKC b isoform was translocated. Accordingly, the decreased availability of cytosolic PKC may contribute to the diminished PKC-dependent responses to CD3-triggered stimulation of human T lymphocytes with aging (Fulop et al., 1995). Immunoblot analysis with isoenzyme specific antibodies showed that T cells from 5 of 11 elderly subjects exhibited selective reductions in PKCa that was ,60% of those in young subjects, whereas the levels of PKCb were comparable to T cells of young subjects (Whisler et al., 1995). No age-related reductions of PKCa or b were observed in B cells. Certain thiol compounds such as 2ME are known to enhance the T cell-dependent immune response of mice in vivo and the proliferation of T cells in vitro. In this process, 2ME appears to potentiate translocation of PKC after T cell activation (Fong and Makinodan, 1989). In T cells from aged human there was less translocation of PKC from the cytosol to the membrane following activation with the anti-CD3 antibody, indicating that abnormal membrane signal transduction pathways play a role in T cell dysfunction associated with human aging (Gupta, 1989).

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It is interesting to note that impaired phosphorylation of PKC is also observed in the brain during senescence (Pascale et al., 1998). Impairment of the Ras pathway Ras is another important signal molecule during T cell activation. Two adapter proteins, Grb2 and Shc, have recently been found in the transmission of activation signals from the stimulated TCR to Ras. Aging leads to a decline in CD3-stimulated phosphorylation of Shc (but not Grb2), and to an increase in CD4-stimulated phosphorylation of Grb2, Shc, and the 30-kDa Grb2-like protein. The differences between T cells from young and old mice suggest that aging may lead to a set of alterations in kinase/substrate coupling that contribute to immune dysfunction in the elderly, and that activation of the Ras pathway might be impaired by aging in T lymphocytes (Ghosh and Miller, 1995). Extracellular signal-regulated kinases (ERK) and c-Jun NH2-terminal kinases (JNK) are molecules belonging to the MAPK family and important for IL-2 production. Grossmann et al. (1995) reported that diminished ERK2 and JNK catalytic activities were commonly detected in T cells from elderly humans stimulated with anti-CD3 mAb OKT3 plus PMA. The induction of JNK activity did not change significantly with age; however, the induction of MAPK and ras activities was significantly less (50 to 65%) in T cells from old rats than in T cells from young rats. In addition, the reductions of ERK2 activation in stimulated T cells from elderly individuals were accompanied by decreased Raf-1 kinase activation, and could be observed without coexisting impairments in JNK activation (Pahlavani et al., 1998). Thus, it is likely that some abnormalities occur in the MAPK activation pathway of T cells from both the elderly human (Whisler et al., 1996a) and the aged mice (Gorgas et al., 1997). Transcription factors Signals received by receptors on the cell membrane eventually activate various transcription factors or their precursors, which regulate transcription of various genes. Activation of AP-1, a heterodimer of c-jun and c-fos, was significantly reduced in PHA-stimulated T cells from the group of elderly subjects when compared to T cells from young subjects. Even though PHA-stimulated T cells from eight of the elderly subjects had pronounced impairments in the activation of AP-1, additional signals provided by costimulation with PMA frequently restored AP-1 activation to more normal levels (Whisler et al., 1993, 1996b). Expression of c-jun mRNA was decreased in peripheral blood lymphocytes from aged humans, when stimulated with PHA (Song et al., 1992). Salkind (1994) examined human T cells exposed to influenza A virus and found the decreased production of proteins Fos and Jun in T cells from elderly persons. NFkB and NFAT are known to play an important role in the expression of various cytokine genes of T cells. Pahlavani et al., (1995) reported the decreased ability of nuclear extracts isolated from T cells from old rats to bind NFAT oligonucleotide. In this respect, activated T cells from elderly donors showed a significant reduction in the induction of NFkB (Trebilcock and Ponnappan, 1996). The proteins coded by c-myc and c-myb protooncogenes are also known to function as DNA binding protein, playing a role in cell proliferation. After PHA stimulation, lymphocytes from old donors showed a decrease of the percentage of lymphocytes expressing c-myb protein (Pieri et al., 1992). Oxidative stress is believed to play an important role in aging processes, and it is interesting to note that transcription factors, NFkB and AP-1, are modulated by oxidative stress (Pahlavani and Vanremmen, 1997; Ginn-Pease and Whisler, 1998).

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CONCLUSION The age-related changes in the immune system mainly occur in T cell-dependent functions. Among various functions of T cells, the most important one is the recognition of antigen by TCR, which can be detected as the proliferative response or cytokine production. The proper T cell response by antigenic stimulation needs costimulation through the CD28 molecule on the cell membrane, and the response is also modulated by signals from cytokine receptors. As the numbers of TCR and cytokine receptors do not change with age in the resting phase of T cells, most investigators assume that the age-related changes may occur in the intracellular signal transduction. So far, there have been accumulating data indicating that a significant decrease occurs in the activity or expression of many signal-related molecules in T cells from aged people or animals after activation, as summarized in Fig. 1. Malfunction of signal-related molecules could be ascribed to either structural change in the molecule itself, downregulation by other molecules, or dysfunction of another molecule upstream. A change in molecular structure may occur by oxidative stress or by a mutation in the corresponding gene. Patterns of tyrosine phosphorylation after antigenic stimulation are different between T cells from young and elderly subjects, some phosphoproteins decreasing and others increasing in T cells from the elderly. There is a possibility that some of these increasing phosphoproteins may downregulate the activity of some signal-related molecules. In this respect, expression of p21 (Waf1) protein is known to increase in T cells from the elderly, playing a role in the suppression of proliferative activity (Hirokawa, 1998). The decreased production of second messengers (IP3 and DAG) can be ascribed to a malfunction of PLCg1, which in turn, is caused by alteration of tyrosine kinase associated with the TCR–CD3 complex. Thus, a T cell clone from old mice can be fully activated to the young level by stimulation with PMA plus ionomycin, which directly stimulates PKC and induces influx of Ca21, respectively. The site of age-related changes in signal pathways may be different for T cell clones and T cell subsets. However, stimulation of splenic T cells freshly prepared from old mice, which are composed of many clones, can be considerably, if not fully, restored to young level by stimulation with PMA plus ionomycin. These results, taken together, suggest that there are common key molecules, abnormalities of which severely influence the cellular functions. For instance, molecules in the early signal pathway following TCR are more susceptible to aging compared with those in the downstream. Abnormalities of receptors themselves are likely to occur during aging processes. Re-expression of TCR after activation is significantly retarded in T cells from the aged mice. Expression of cytokine receptors is much slower in T cells from the aged than in those from young mice. CD28-negative T cells increase in proportion with age. In any event, further studies will be required to precisely determine key molecules that are responsible for impairment of signal transduction of T cells. We expect that the information about key molecules in the aging process of T cells may lead to exploration of methods to restore the function of T cells and the immune system of the aged individuals. REFERENCES BECKMAN, I., SHEPHERD, K., FIRGAIRA, F., and AHERN, M. Age-related defects in CD2 receptor-induced activation in human T-cell subsets. Immunology 86, 533–536, 1995. BENDER, B.S. and TALLMAN, E. The heterogeneity of the age-related decline in immune response: Impairment in delayed-type hypersensitivity and cytotoxic T-lymphocyte activity occur independently. Exp. Geront. 27, 347–354, 1992. CHAKRAVARTI, B., CHAKRAVARTI, D.N., DEVECIS, J., SESHI, B., Abraham, G.N. Effect of age on mitogen

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