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Apoptosis represents the main mechanism involved in the intrathymic cell selection. The involution and atrophy of the thymic gland during aging has been associated with an altered representation of thymocyte subsets and particularly of CD4 ؉ CD8 ؉ double-positive (DP) thymocytes, i.e., the cell population mainly involved in thymocyte selection. The aim of the present study was to evaluate the responsiveness of thymocytes from old mice to factors regulating the apoptotic cell death, such as antibodies to CD3/T cell receptor complex, zinc, and dexamethasone (DEX).Balb/c mice were used at the ages of 2 months (young), 21-22 months (old), and 24-26 months (very old). Thymocytes from these mice were incubated overnight with anti-CD3 monoclonal antibody (8 g/ml), Zn 2؉ (150 M), or DEX (10 ؊7 M) and then analysed for number of apoptotic nuclei, cell cycle phase, and phenotype by flow cytometry.A significant decrease of both the total number and the proportion of DP thymocytes was present in old and very old mice in comparison with young animals. Antibodies to CD3 antigen induced thymocyte apoptosis in both young and old mice. The stimulation of the CD3/TCR complex was more effective in giving apoptosis in very-old than in old and young mice. An impairment of the inhibiting effect of zinc on apoptosis induced by either serum deprivation or DEX was found in old and very old mice, whereas zinc was less effective in inhibiting CD3-induced apoptosis only in very old animals. Reduced DEX-induced apoptosis was also present in old age; this effect was more evident in very old than in old mice. Thymocyte apoptosis in old mice required protein synthesis being blocked with cycloheximide. Apoptosis was exerted on thymocytes in a specific cell cycle phase, i.e., on G0/G1 phase cells. Anti-CD3 antibodies, Zn 2؉ , or DEX regulated apoptosis by modulating the proportion of DP thymocytes.The results demonstrate an altered in vitro responsiveness of thymocytes from old and very old mice to factors regulating apoptosis and suggest further investigations to determine if this altered responsiveness is associated with increased apoptosis of thymocyte populations occurring with increasing age. Cytometry 32:1-8, 1998. 1998 Wiley-Liss, Inc.Key terms: flow cytometry; thymocyte apoptosis; cell cycle; monoclonal antibody; aging Experimental and clinical evidence has accumulated in favour of the thymus as a critical organ in mammalian immunologic aging (19,40). In fact, the decline of immunocompetence in aging from alterations in number or function of lymphoid populations is closely linked to the involution and atrophy of the thymus gland, which begins after the onset of puberty and proceeds with age (16,19,45). The proportion and number of lymphocytes in the thymus decrease with age, as does thymus cellularity. This decrease has been reported to occur in both cortex and medulla of the thymus, with a slight faster rate of involution at the cortical level (29,38).

Apoptosis represents the main mechanism involved in the intrathymic cell selection. The involution and atrophy of the thymic gland during aging has been associated with an altered representation of thymocyte subsets and particularly of CD4+CD8+ double‐positive (DP) thymocytes, i.e., the cell population mainly involved in thymocyte selection. The aim of the present study was to evaluate the responsiveness of thymocytes from old mice to factors regulating the apoptotic cell death, such as antibodies to CD3/T cell receptor complex, zinc, and dexamethasone (DEX). Balb/c mice were used at the ages of 2 months (young), 21–22 months (old), and 24–26 months (very old). Thymocytes from these mice were incubated overnight with anti‐CD3 monoclonal antibody (8 μg/ml), Zn2+ (150 μM), or DEX (10−7 M) and then analysed for number of apoptotic nuclei, cell cycle phase, and phenotype by flow cytometry. A significant decrease of both the total number and the proportion of DP thymocytes was present in old and very old mice in comparison with young animals. Antibodies to CD3 antigen induced thymocyte apoptosis in both young and old mice. The stimulation of the CD3/TCR complex was more effective in giving apoptosis in very‐old than in old and young mice. An impairment of the inhibiting effect of zinc on apoptosis induced by either serum deprivation or DEX was found in old and very old mice, whereas zinc was less effective in inhibiting CD3‐induced apoptosis only in very old animals. Reduced DEX‐induced apoptosis was also present in old age; this effect was more evident in very old than in old mice. Thymocyte apoptosis in old mice required protein synthesis being blocked with cycloheximide. Apoptosis was exerted on thymocytes in a specific cell cycle phase, i.e., on G0/G1 phase cells. Anti‐CD3 antibodies, Zn2+, or DEX regulated apoptosis by modulating the proportion of DP thymocytes. The results demonstrate an altered in vitro responsiveness of thymocytes from old and very old mice to factors regulating apoptosis and suggest further investigations to determine if this altered responsiveness is associated with increased apoptosis of thymocyte populations occurring with increasing age. Cytometry 32:1–8, 1998. © 1998 Wiley‐Liss, Inc.

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