]>MAD1887S0047-6374(97)01887-310.1016/S0047-6374(97)01887-3Elsevier Science Ireland LtdFig. 1Proliferative responses of PBMC from 14 centenarians, 10 middle-aged and 16 young controls to soluble anti-CD3 mAb (25 ng/ml) or PMA (5 ng/ml).Fig. 2Proliferative responses of purified T cells from 14 centenarians, 10 middle-aged and 16 young controls to PMA (5 ng/ml); PMA added to immobilized anti-CD28 mAb, immobilized anti-CD3 mAb or to coimmobilized anti-CD3 plus anti-CD28 mAb.Table 1Cytofluorimetric analysis of T lymphocytes from centenarian, middle-aged and young subjectsCentenariansMiddle agedYoung adultsMean age and number of subjects101.1±0.4 (10)63.5±1.9 (17)28.8±0.9 (24)% of CD28+ among T cells50.5±3.5**77.3±3.8**90.25±1.4Number of CD28+ T cells per μl583±47**1138±70**1558±47CD28 median fluorescence channel in linear scale385±13.9358±21372±18Data are reported as mean values±S.E.M.PBMC were double stained with FITC-conjugated mAb anti-CD3 and PE-conjugated mAb anti-CD28 as described in Section 2.Analysis of CD28 expression was obtained by Lysis II® software.Statistical analysis was done by using the ANOVA Fischer test.**P<0.01 vs. either of the two other groups.T lymphocyte proliferative capability to defined stimuli and costimulatory CD28 pathway is not impaired in healthy centenariansPaoloSansonia*FrancescoFagnoniaRosannaVescoviniaMarcoMazzolaaVincenzoBriantiaGiovanniBolognaaEnricaNigroaGiampaoloLavagettoaAndreaCossarizzabDanielaMontibClaudioFranceschibcMarioPasseriaaIstituto di Clinica Medica Generale e Terapia Medica, University of Parma, via Gramsci 14, Parma 43 100, ItalybDipartimento di Scienze Biomediche, Sezione di Patologia Generale, University of Modena, via Campi 287, Modena 41 100, ItalycINRCA, via Birarelli, 8, 60 100 Ancona, Italy*Corresponding author. Tel.: +39 521 290783/4; fax: +39 521 290776.AbstractIt is generally assumed that T cell proliferation is impaired in aged individuals. We report data on the proliferative capability of peripheral blood mononuclear cells (PBMC) and T lymphocytes from 40 healthy people of different ages, (19–107 years), including 14 centenarians, to defined mitogenic stimuli. We observed no age-related proliferative impairment both in PBMC and in purified T cells stimulated by anti-CD3 mAb or phorbol myristate acetate (PMA). Furthermore, T cells stimulated by anti-CD3 mAb or PMA and costimulated by CD28 mAb did not proliferate differently among young, middle aged subjects and centenarians. Thus, short term T cell proliferation is not affected even at extreme age when well defined stimuli are used on cells deriving from carefully selected healthy subjects.KeywordsCentenariansT cellsCD28Proliferation1IntroductionAn old tenet in medicine is that aging is accompanied by a deterioration of the immune system that could contribute to increased risk of morbidity and mortality [1–4]However, the study of carefully selected healthy aged subjects showed that most immune functions are well preserved even in advanced age [5, 6]. We have shown that several immune responses are well preserved in healthy centenarians in comparison to middle-aged and young subjects[7, 8].A very important function which has been suggested to deteriorate with age and to play a major role in the aging process is the capability of cells from aged subjects to respond to mitogenic stimuli and, consequently, to undergo cell proliferation [9]. A defect in this function is very important for immune cells, whose capability to undergo clonal expansion is critical to set up an effective response to antigenic stimuli [10]. Accordingly, we thought it worthwhile to assess the proliferative capability of peripheral blood mononuclear cells (PBMC) and purified T lymphocytes from healthy centenarians in comparison to middle-aged and young subjects. Our results demonstrate that this function is well preserved when defined stimuli, such as anti-CD3 mAb or phorbol 12-myristate acetate (PMA), are used. This conclusion is further supported by experiments showing that the costimulatory pathway via the CD28 molecule is well preserved in centenarians.2Materials and methods2.1Subjects.We studied a total of 14 centenarians with a mean age of 100.8±0.3 years (range, 100–107 years), 3 males and 11 females. All subjects were in relatively good clinical condition without relevant acute or chronic disease affecting the immune system and mentally competent to give informed consent. In particular, none of the subjects had cancer, were suffering from serious cardiac, brain or kidney disease or taking drugs known to affect the immune system. Along with a complete social and medical history, we performed a physical examination. As control groups we studied 10 healthy middle-aged subjects with a mean age of 58.2±2.1 years (range, 48–68 years) and 16 healthy young donors with a mean age of 28.1±0.6 years (range, 19–36 years). All these control subjects were selected according to the SENIEUR protocol [5, 6].2.2Monoclonal antibodies.MAb to CD3 (OKT3) was generously provided by Dr E. Engleman, Stanford University (Stanford, CA); anti-CD8 conjugated with fluorescein (FITC), anti-CD28 conjugated with phycoerythrin (PE) and isotype-matched control mAb were purchased from Becton Dickinson (San José, CA).2.3Cell preparation and enrichment of T lymphocytes.PBMC were obtained by Ficoll-Hypaque gradient centrifugation from freshly drawn venous blood collected around 08:00 from a centenarian, a middle-aged and a young control on the same day. After washings with PBS, the cells were suspended in RPMI-1640 with 10% AB serum, 2mM l-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin, hereafter referred to as complete medium. PBMC were fractionated into T and non-T cells by a single step rosetting method [11]. T cells were separated from sheep red blood cells by hypotonic lysis of the latter. T cells were further depleted of monocytes by adherence (1 h) to plastic. The purity of T cells preparation was always >95% as assessed by cytofluorimetric analysis.2.4Proliferation assays.All proliferation assays were performed in round-bottomed microtitre wells in a final volume of 0.2 ml complete medium. Stimulation of 1×105 PBMC with soluble anti-CD3 mAb (25 ng/ml) or 5 ng/ml PMA was carried out for 3 days at 37°C in 6% carbon dioxide–air. Purified T cells were stimulated with immobilized mAb as follows: 100 μl anti-CD3 (0.1 μg/ml) and/or 100 μl anti-CD28 (5 μg/ml), diluted in PBS, were placed in 96 wells microtiter plates and incubated at room temperature overnight and then washed with PBS. T cells were challenged with several stimuli or a combination of them: immobilized anti-CD3; immobilized anti-CD3+immobilized anti-CD28; 5 ng/ml PMA; PMA+immobilized anti-CD28 as specified in the figure legends. Assays were performed in triplicate and 0.5 μCi [3H]thymidine ([3H]TdR) was added to each well 6 h before cell harvesting on glass fiber filter paper. Uptake of [3H]TdR was measured in a liquid scintillation counter and the results expressed as the mean counts per min±S.E.M. (cpm±S.E.M.).2.5Cytofluorimetric analysis.Cytofluorimetric analysis was performed on PBMC following standard methods [12]. Briefly, 1×106 PBMC were incubated with 1 μg anti-CD8 FITC and anti-CD28 PE for 20 min at 4°C, washed with PBS and suspended in 200 μl PBS until analysis. Two colors FACS analysis was performed on a FACScan cytofluorimeter (Becton-Dickinson, CA) as previously described [12]. The expression per cell of CD28 molecule was calculated by flow cytometry using PE-conjugated anti-CD28 mAb, considering the mean fluorescence channel of each histogram, as described [13].2.6Statistical analysis.Statistical analysis was performed by ANOVA and Fisher test by SPSS for Windows® software. P<0.05 was considered significant.3Results3.1Proliferative responsiveness of PBMC and purified T cells.When PBMC were stimulated by defined mitogenic stimuli, such as PMA or anti-CD3 mAb, no significant difference concerning the proliferative capability was observed among cells from young, middle-aged and centenarians (Fig. 1). PBMC are mainly composed of T and B lymphocytes, NK cells and accessory cells such as monocytes. It is well known that optimal T cell responsiveness requires a primary and a costimulatory signal that usually is delivered by accessory antigen presenting cells through the B7 molecule which is capable of interacting with the CD28 molecule on the T cell membrane [14]. Accordingly, purified T cells from young, middle aged and centenarians were stimulated by the above mentioned primary stimuli (PMA or anti-CD3 mAb) and costimulated by anti-CD28 mAb in order to activate the most important costimulatory pathway in T cells [15]. Fig. 2 shows that costimulation via anti-CD28 mAb significantly increased T cell responsiveness to PMA or anti-CD3 mAb in cells from all groups including centenarians and again no significant age-related difference was found.3.2CD28 expression.Since the target of anti-CD28 mAb is the CD28 molecule on the T cell membrane we performed a cytofluorimetric analysis of resting lympocytes in order to study the expression of this molecule on T lymphocytes from centenarians, middle-aged and young controls.The results indicate that the costimulatory capability of anti-CD28 mAb is not likely to be related to the percentage of CD28+ cells in culture. Indeed Table 1 shows that the percentage of CD28+ cells is significantly reduced in centenarians, whose cells, however, responded well to costimulation with anti-CD28 in the presence of PMA or anti-CD3 mAb (Fig. 2). Table 1 also shows that, notwithstanding the decrease in the percentage of CD28+ lymphocytes, the expression of CD28 molecules per cell was, however, unchanged.4DiscussionPrevious studies indicate that a profound remodelling of the immune system occurs with age [7]. In particular we have reported that, in the peripheral blood of aged people including centenarians, there is a progressive increase in serum level of IgA, IgG1, IgG2, IgG3 but not of IgM or IgG4 [16], a decrease in absolute number of T lymphocytes (CD3+) involving both CD4+ and CD8+ subsets, a marked decrease of B lymphocytes and an increase of cells with natural killer (NK) markers [8, 17]. We have also found that healthy elderly and centenarians are almost free of organ-specific autoantibodies [18, 19]and equipped with very well preserved cytotoxic activities (NK, anti-CD16 and anti-CD3 redirected killing activities) [8, 17, 20].An important and extensively studied immune function is lymphocyte capability to undergo cell proliferation. Several reports indicate that this function is impaired with age [1, 2]. The reason for this defect has been variably referred to among T cell subsets, an imbalance [21], a reduced proliferation of CD8+ T cells [22], a failure to produce IL-2 [23]or to a decreased number of cells capable of completeing the S phase of the cell cycle [24]. However, most of the previous studies refer to the capability of PBMC to proliferate when stimulated with PHA, a mitogen used to assess such a function since 1960 but whose fine mechanism is not completely understood [25]. The available data indicate that PHA induces activation of T cells in a complex way through cross-linking of several surface structures, including CD2, CD3, CD5 molecules [26, 27]and perhaps others. Thus, we thought it worthwhile to study the proliferative capability of either PBMC or purified T cells from people of different ages using more defined stimuli acting at different levels of the signal transduction cell machinery, i.e. plasma-membrane (anti-CD3) or transmembrane (PMA). When this approach was used no proliferative defect was evident with both stimuli in cells from any group, including people who are close to the maximum life span such as subjects over 100 years of age. These data are in accord with earlier reports in unseparated PBMC from subjects aged between 70 and 82 years [28, 29].We tried to further dissect the pathways known to be involved in lymphocyte proliferation and particularly the main costimulatory T-cell pathway via the CD28 molecule. To this end we used anti-CD28 mAb to act as surrogate for this most important contribution by accessory cells to T cell activation [14]. This system has the advantage of optimizing the lymphocyte proliferative conditions, indeed the ligation of CD28 molecules increases the expression of IL-2 receptors and IL-2 availability [15, 30, 31]which, in turn, are thought to be defective in lymphocyte cultures from elderly subjects [23, 32]. Under these conditions, costimulation via CD28 significantly increased T lymphocyte proliferation in middle-aged and young controls, as well as in centenarians, and this phenomenon was of the same extent in the three groups.According to the two signal theory, T lymphocyte activation derives from MHC-peptide specific engagement of TCR-CD3 and contestual crosslinking of non antigen-specific accessory molecules such as CD28 [33, 34]. Our data indicates that both CD3 and CD28 pathways are well preserved in T cells from aged people including centenarians. Our results suggest that the cell machinery responsible for the transduction of primary and costimulatory signals is functioning well even in cells from these subjects of very advanced age. The results presented are in apparent contrast with the data in the literature indicating that lymphocytes from elderly people show a proliferative defect when complex stimuli such as PHA [2]or alloantigens are used [35]. The defective response to PHA could be related to the complexity of this stimulus (proliferation of different T cell subsets? Activation-induced cell death? Induction of cell nonresponsiveness?).The results shown here suggest that, when cells from carefully selected aged subjects are used and stimulated with well defined stimuli T lymphocyte proliferation is not impaired as previously thought and that some important activation pathways, such as those involving CD3, are well preserved into the last decades of life. Furthermore, the efficient costimulation through CD28 molecules in PMA and in anti-CD3 stimulated cultures indicate that the costimulation through the CD28 pathway is not impaired in centenarians despite the reduced number of CD28 positive cells with age.About 1/3 of centenarians, despite their very advanced age, are still in good mental and physical condition. These subjects are the best example of successful aging, being free of the major age-related diseases. Therefore, healthy centenarians are a model of human longevity and constitute a very select group of exceptional individuals. The current observation of a well preserved T cell proliferative capability involved in adaptive immune responses, together with previous observations concerning well preserved innate immunity, suggests that such a well equipped immune system may contribute to longevity in human and, on the other hand, indicate that the immune system of healthy centenarians is still apparently capable of coping with infectious diseases.In summary, we suggest that a generalized age-related defect in the cell proliferative machinery can be excluded and that important activation pathways are intact in lymphocytes even in the last decade of life. The data presented here fit the hypothesis that aging is not characterized by unidirectional deterioration but by a complex remodelling of immune functions [7, 36].AcknowledgementsThis work was supported by the National Research Council (CNR) target project `Ageing', M.U.R.S.T. 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