Recent studies (1, 2 ) with thymus-dependent antigens have conclusively confirmed earlier demonstrations (3, 4) that there is a dramatic decline in humoral immune competence during senescence. Systematic studies to determine whether a comparable decline in cell-mediated immunity occurs are needed, as previous studies are not in agreement [for review see Ref.(5) 1. More recently it has been postulated, and considerable interest has been generated in the concept, that agerelated failure of immune surveillance mechanisms may play a prominent role in carcinogenesis and other age-dependent disease processes (6-9). Conclusive demonstration of a decline in cell-mediated immunity is essential if this concept is to remain tenable. I t is now clearly established that thymusdependent lymphocytes (T cells) are an absolute requirement of cell-mediated immunity (10-12). I t is generally accepted that phytohemagglutinin (PHA) selectively stimulates T cells (13-15), and therefore PHA stimulation of lymphocytes is commonly used as a measure of cell-mediated immunity (16, 17). Thus, it seems apparent that the in vitro blastogenic response of mouse spleen cells to PHA should be useful in identifying age-related immunodeficiencies that might exist in the T-cell population of the spleen, although it is recognized that PHA responsiveness at best measures only one parameter of cellmediated immunity.Materials and Methods. Mice used for these experiments were a long-lived hybrid (C57BL/Cum X C3H anf/Cum $)F1(hereafter referred to as BC3F1) with a mean life-span of 31 months and a maximum life-span of 45 months and the shorter-lived BALB/c strain with a mean life-span of approximately 25 months. Spleens were removed under sterile conditions. Only spleens of normal size and free of pathology were used. Subsequent his tological examinat ion proved the spleens to be pathology free. Cell suspensions from individual spleens were prepared in Hanks' balanced salt solution, counted in a hemacytometer, and adjusted to contain 10 X lo6 leucocytes/ml. Routinely, the culture media consisted of 89% RPMI-1630 (Grand Island Biological Co., N. Y.), 10% fetal calf serum, and 1 % penicillin-streptomycin (5000 units penicillin and 5000 pg streptomycin/ml) . Five replicate cultures were prepared for each spleen cell suspension in screw-cap Falcon flasks (30 ml capacity with a bottom area of approximately 5.25 cm2) by adding 2.5 X lo6 spleen cells (0.25 ml) to each flask, which contained 3.75 ml of the complete culture medium. Cultures ~were loosely capped and incubated at 37" in a 5% C 0 2 atmosphere at 80% humidity.was reconstituted with sterile distilled water
The late effects of various immunosuppressive insults on cell-mediated immunity in mice were studied in an attempt to assess the role of immune surveillance in the aging process. Results were obtained using susceptibility to allogeneic tumor cell challenge, graft-versus-host reaction (GVHR), blastogenic response to PHA, a thymus derived T cell-specific plant mitogen, and cytolytic activity against allogeneic tumor cells as measures of immunologic activity. In vivo studies late in life show that resistance to allogeneic tumor cells is significantly decreased in thymectomized mice, whereas those treated with cortisone, cyclophosphamide and sublethal X-ray remain unchanged. Spleen cells from only the thymectomized and the sublethally irradiated mice show reduced activity in the GVHR. No difference is seen in the activity of bone marrow cells. Results consistent with these findings were obtained in in vitro studies. Thus spleen cells from thymectomized or sublethally irradiated mice show decreased activity is response to PHA, whereas no change is seen in spleen cells from other treated groups. Hence, surgical and physical insults are more likely to induce long-lasting immunosuppression in those immunocompetent tissues whose activity normally diminishes with advancing age. Furthermore, the degree of immunosuppression seen in this study is not of the order of magnitude that one could reasonably predict a significant decrease in mean life-span.
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