Proliferative and cytotoxic immune functions in aging mice. III. Exogenous interleukin-2 rich supernatant only partially restores alloreactivity in vitro

Mechanisms of Ageing and Development, 31 (1985) 103-113 Elsevier Scientific Publishers Ireland Ltd.

i 03

PROLIFERATIVE AND CYTOTOXIC IMMUNE FUNCTIONS IN AGING MICE. IlI. EXOGENOUS INTERLEUKIN-2 RICH SUPERNATANT ONLY PARTIALLY RESTORES ALLOREACTIVITY I N VITRO*

SUSAN R.S. GOTTESMANa, ROY L. WALFORD b and G. JEANETTE THORBECKEa aDepartment of Pathology, New York University School of Medicine, New York, N Y 10016 and bl~partraent of. Pathology, School of Medicine, University of California, Los Angeles, CA 90024

(U.S.A.) (Received November 20th, 1984) SUMMARY The ability of exogenous intefleukin-2 (IL-2) rich supematant to restore the defective T cell mediated immune functions of spleen cells from aged C57BL/6 mice was analyzed. Addition of IL-2 rich supematant to allogeneic mixed lymphocyte cultures (MLC) resulted in an increase .in the proliferative response of spleen cells from both young and old mice. The MLC response of cells from old mice was, however, not restored to the level of proliferation seen with splenocytes from young animals. In studying the generation of specific T cell suppressor function, it was found that IL-2 rich supematant enhanced this function only for spleen cells from those aged animals which demonstrated a defective response in its absence. The response of these mice was thereby restored to the normal level. The response of cells from young control animals and aged mice with normal suppresssor activity was not affected by the addition of IL-2 rich supematant. We conclude that decreased IL-2 production constitutes a functionally important aspect, but is by no means the only defect in the immune response of aged mice. The results also suggest that responsiveness to IL-2 is less affected by age than lymphokine production.

Key words: Cell mediated immunity; Interleukin-2; Suppressor cells; Mixed lymphocyte

cultures; Aging

*This work was supported by US Public Health Service Research Grants NIH AG-04980 and AG04860, DHHS. Address all correspondence to: Dr. Susan R.S. Gottesman, Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, U.S.A. Abbreviations: Ii,-2, interleukin-2; MLC, mixed lymphocyte culture; 136,C57BL/6;B6D2, (C57BL/6 X DBA/2)Ft; PHA, phytohemagglutinin.

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104 INTRODUCTION In aged individuals, evidence of ineffective cooperation by cells of the immune system is suggested by observations of severely deficient immune functions with relatively little variation in cell numbers [1]. Our previous studies demonstrated declines in the proliferative, cytotoxic, and specific suppressor T cell responses to foreign histocompatibflity antigens by spleen cells from aged as compared to young C57B1/6 (B6) mice [2,3]. The proliferative response showed an earlier and more severe decline with age than the other two (functional) assays. The immune system communicates through the production of soluble mediators (lymphokines) which stimulate cells by means of surface receptors [4]. Numerous investigators have demonstrated a decline in lymphokine, particularly interleukin-2 (IL-2), production with .age [5-12]. There is disagreement, however, on the ability of exogenous IL-2 to restore various immune functions to young adult levels. Whereas some investigators have reported complete restoration of immune responses by supplementation with IL-2 preparations [6,8,10,13], others have maintained that with advanced age, there e x i t deficiencies in both the ability to produce and the ability to respond to this lymphokine [5,7,11,12,14]. In the present study we have reinvestigated the relative importance of defective IL-2 production in the deficiencies of T cell immune responses of aged mice. The results to be reported demonstrate that, whereas exogenous IL-2 rich supematant is able to induce similar percent increases in the proliferative responses of young and aged animals, such supematant is unable to restore the absolute values of the severely deficient aged mouse T cells to the level of the young. In the generation of a specific suppressor cell response, IL-2 rich supematant is again able to improve the response of those aged animals tested which had deficient supppressor activity. As this functional activity is less severely affected by age, an improvement in the response brings it to a level indistinguishable from the young controls. We conclude that supplementation with IL-2 leads to significant improvement of immune functions but that aged mice have additional deficiencies which cannot be overcome by adding IL-2. MATERIALS AND METHODS Mice

Male mice of the inbred strains C57B1/6NCrlBR (B6, Charles River Labs., North Wilmington, MA), DBA/2 and (C57BL/6 X DBA/2)F 1 (B6D2) (Jackson Labs., Bar Harbor, MN) were used. B6 mice of 4 - 8 months were considered young adults, 14--20 months middle-aged and 2 3 - 3 0 months aged. Mice were discarded if, at the time of death, signs of tumors or infections were found. lnterleukin-2 (11,-2) - preparation and quantitation

IL-2 was produced by the T cell line, LBRM-33. Cells were grown to confluence and then stimulated with phytohemagglutinin (PHA) (4/ag/ml) for 4 h. Cells were then washed

105 and incubated in medium without PHA for 20 h before the supematant was harvested. IL-2 was assayed using an IL-2-dependent cell line CTLL.All (courtesy of Dr. M.A. Palla. dine, Genentech, South San Francisco, CA). The effect of serial d.flutions of supernatant fluids on [aH]thymidine incorporation by 5 X 10 a cells/well in microtiter plates was determined and the amount of IL-2 was expressed in U/ml after probit analysis [ 13]. One preparation of IL-2 rich supematant (720 U/ml) was used for the entire series ofexperi. ments.

Mixed lymphocyte cultures (MLCJ (modification of Refs. 16 and 17] Pooled spleen cells from young adult (4-8 months) mice were irradiated (1500 R, laTCesium Source, Isomedix, Parsippany, N J) and used as stimulators (2 X l0 s cells/well of B6, syngeneic, B6D2, semi.allogeneic, or DBA/2, allogeneic) while spleen cells from individual B6 mice of various ages constituted the responder populations (0.5-2.5 X 10 s cells/well). When B6 mice of different ages were used as stimulator cells, animals were tested individually. The cells were incubated in flat bottom Linbro microtest plates (No. 76-003-05) in a total of 0.2 ml culture medium [Eagle's minimal essential medium, 2 mM L-glutamine, i00 U/ml penicillin-streptomycin, 1% non-essential amino acids, 10% heatinactivated fetal calf serum and 2 X lO -s M 2-mercaptoethanol (Eastman Co., Rochester, NY)]. Six replicates were set up for each cell dose. In some experiments 10/~l of the IL-2 preparation (7.2 U or 5% v/v) or lO gl of control medium were added to wells on day 0. Total volume/well was kept constant at 0.2 ml. Tritiatod thymidine (New England Nuclear Co., 2.0 Ci/mmol, 1 #Ci/weU) was added for the last 8 h of a 4 or 5 day incubation period and cultures were harvested. Background proliferation was obtained using irradiated young syngeneic cells as stimulators. Alternately, cultures were set up using 12 X 106 B6 responder cells and 12 X l06 irradiated DBA/2 stimulator cells in a total of 6 mi. Aliquots were then taken every 24 h and labeled with tritiated thymidine for 14 h thus generating a kinetics curve of the proliferative response [2].

Generation and assay of specific suppressor cells [modification of Ref. 18] B6 cells (2 X 106/ml) were stimulated in test medium with irradiated (1500 R) DBA/2 stimulator cells (4 X 106/ml) for 4 days in 24 well tissue culture plates (Costar, Cambridge, MA). IL-2 rich supematant (72 U or 5% v/v) or medium was added to the generation cultures. Total volume was kept constant at 2 ml/well. Cells were harvested, washed, counted and irradiated with 1000 R before being added to the fresh assay MLC. The irradiated "suppressor cells" were added in varying doses to the fresh MLC in which spleen cells from young B6 mice served as responders, and the total volume/well was kept constant at 0.2 ml/well. Cultures were harvested after a 5-day incubation.

Statistical analysis Comparisons between responses of the same dose of cells from young and aged mice were analyzed using the two-tailed Student's t-test for paired comparisons. Degrees of

106

freedom were corrected based on f-tests results. Statistical evaluation of the significance of differences between young and old responder cells were determined for cell doses on the linear part of the dose response curve. EXPERIMENTAL RESULTS

Comparison of responses to dlogeneic and semi-allogeneic stimulator cells Irradiated fully allogeneic stimulator spleen cells contribute to the proliferative response in the MLC through the mechanism of back stimulation and lymphokine production. Therefore, we compared the ability of spleen cells from mice of different ages to respond in the MLC to fully allogeneic and semi-allogeneic stimulators. Figure 1 shows a representative experiment of the response of two middle aged and one young B6 mouse to B6D2 stimulator cells (Fig. 1A) and to DBA/2 stimulator cells (Fig. 1B). All mice had a greater response to the fully allogeneic than to the semi-allogeneic cells. The difference between young and middle aged responder cells was significant with both stimulator cells, although greater with the fully allogeneic stimulators, i.e. in the probable presence of lymphokine production through back stimulation. This same pattern of response was found in comparisons of four 3-month-old and seven middle age and aged animals (18 and 23 months).

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Fig. 1. Comparison of the proliferative response of spleen cells (2 × 106/ml) from 1 young (3 months) and 2 middle aged (18 months) B6 mice to irradiated semi-aUogeneic (B6D2, panel A) and fully aUogeneic (DBA/2, panel B) stimulators (2 × 106/ml). AUquots were taken every 24 h and labeled with tritiated thymidine for 14 h. Each point represents the average of 6 replicates. *P < 0.01, +P < 0.05 compared to response of young controls at same time point, yg, young; mr, middle aged; and ma, middle aged.

107 To control for the possibility that cells from older cause back stimulation, spleen cells from young (3 were compared for their ability to act as stimulators The aged mouse spleen cells showed an equivalent,

mice might have a reduced ability to months) and old (23 months) mice in the fully allogeneic MLC (Fig. 2). if not greater, ability to stimulate.

Effect of lymphokines on MLC responses from young and aged mice We then looked at the ability of excess exogenous IL-2 rich supematant to increase proliferative responses of aged mouse ceils and possibly restore them to the response level of cells from young mice. Over a series of experiments testing six aged ( 2 4 - 2 6 months) and four young ( 5 - 6 months) mice, the percent increase in proliferation caused by addition o f 5% of the supematant (7.2 U IL-2) was similar for young and old mice (Table I). Although this similar relative increase suggests that IL-2 might be able to restore the response of old mice to the level of the young, this was not the case. Even in experiments in which the percent increase for the aged animals was higher than for the young, the absolute response of the aged was still not restored to young adult levels (Fig. 3). The amount of IL-2 added was not the limiting factor, since doubling the amount o f IL-2 rich supematant added to the cultures was without further effect on the response o f the aged.

Effect of lymphokines on suppressor cell generation from young and aged mice IL-2 rich supematant was added to suppressor cell generation cultures to examine if

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Fig. 2. Proliferative response of DBA/2 spleen cells to irradiated B6 stimulator spleen cells from young (3 months) and aged (23 months) mice. DBA[2 responder cell doses from 0.5 to 2.5 × 10s cells/wen were stimulated for 5 days with 2 × 10 s cells/well from one young or one old B6 mouse. Each point represents the average [3H] thymidine uptake for six replicates ± S.E.

108 TABLE I AVERAGE PERCENT INCREASE OF MLC UPON ADDITION OF IL-2 RICH SUPERNATANTb

Responder cell dose

Young adult

(x lO-~)a

(~ + S.E.)C

Aged (~ ±S.E.)¢

0.5 1.0 1.5 2.0 2.5

281 +77 136 +35 1!7 -+39 48 + 22 27 + 8

444 +111 182 +99 151 +58 110 +43 93 + 24

aMLC assays were set up using various doses (0.5-2.5 X l0 s) of responder spleen cells from young and aged B6 mice and a constant number (2 X 10 s) of ir*adiated DBA/2 or B6 stimulator spleen cells. IL-2 bprichsupernatant (5%) or medium was added on day 0 of culture. ercent increase o f proliferative response was calculated after subtraction of the background stimulation by syngeneic B6 stimulator cells with and without added IL-2 rich supernatant. CAve*ageof results of 4 young (5-6 months) and 6 aged (24-26 months) animals tested. No significant difference was found between the two groups of mice in the average percent increase with addition of IL-2 rich supernatant.

the defective response of aged animals could be corrected. Of the five old (26 months) mice assayed, three generated significantly decreased suppressor cell activity without added lymphokine, in comparison to the young controls (P < 0.01 for each o f these experiments). This percentage o f old mice demonstrating a deficient suppressor cell response was similar to that found in a previously tested group of 22 aged animals [3]. It was also shown in these previous studies that addition o f IL-2 rich supematant to the assay cultures o f suppressor cell activity failed to alter the degree o f suppression obtained. The present results show, however, that when IL-2 rich supematant (5% v/v) was added at the beginning of the cultures, it was able to increase the generation o f suppressor cells in cultures with cells from those aged mice that showed a deficient response (Table II). Exogenous lymphokines increased the percent suppression obtained with all the aged mice tested by 16.3% -+ 5.7 (n = 5) (using a suppressor cell test dose of 5 X i0 a cells/well) and by 14.2% +- 1.9 (n = 3) (using a 104 suppressor cell test dose/well). In considering separately the three aged animals which had decreased suppressor generation from the two which had control levels, the former group had a 22.7% and the latter group a 6.1% mean lymphokine induced increase in suppression (using a 5 X 10 a suppressor cell test dose/well). The exogenous lymphokines were without effect when added to the generation o f suppressor cells by spleen cells from young control mice. In the representative experiment shown in Fig. 4, there was no significant effect of IL-2 rich supematant on suppressor cell generation by spleen cells from a young mouse (panel A) or from a non-defective aged mouse (panel B). A second aged mouse (panel C), assayed simultaneously, showed significant improvement o f suppressor cell generation with IL-2 rich supematant in the cultures. This animal was defective in suppressor cell generation in the absence of the added lymphokines, as judged by the previously discussed criteria [3].

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Fig, 3. Effect of addition of exogenous IL-2 rich supernatant (5%) on the proliferative response of spleen ceils of young and aged mice. Spleen cells (0.5 -2.0 X 105/well) from one young (5 months) and two old (24 months) mice assayed individually were incubated for 4 days with DBA/2 stimulator cells (2 X 10 s ceLls/well), with and without IL-2 rich supernatant, For both old mice, significantly less proliferation was generated, even with IL-2 rich supernatant added, than the young mouse without added supernatant. (Old t + IL-2 rich supernatant compared with young: P < 0.05 for 1.0 X 105 cells/ well; P < 0.01 for 1.5-2.0 X 10 s cells/well. Old= + IL-2 rich supernatant compared with young: P < 0.01 for all cell doses).

TABLE II INCREASE IN SUPPRESSOR CELL GENERATION IN THE PRESENCE OF IL-2 RICH SUPERNATANT

Donor o[ spleen cells a

Young mice Aged mice

Increase in % suppression with IL-2 at suppressor cell dose/ well of b 5 XlO s

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0,8 _+0.1 (2) 14.2 -+ 1.9 (3)

aSuppressor cell generation cultures contained young or aged B6 and irradiated DBA/2 spleen cells with and without IL-2 rich supernatant. bT-irradiated (1000 R) before addition to fresh assay MLC between young B6 (1.5 × 105 ceUs/well) and irradiated DBA/2 spleen cells. CDffference between percent suppression generated with and without IL-2. Numbers of individual mice tested given in parentheses.

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Fig. 4. Effect of IL-2 rich supernatant on the generation of specific suppressor cells by spleen cells from mice of various ages. Suppressor cells were generated by spleen cells from young (panel A, 3 months) and old (panel B and C, 26 months) 136 mice by incubation for 4 days with irradiated DBA/2 cells, in the presence or absence of IL-2 enriched supematants. Suppressor cells were irradiated (1000 R) before being added in varying doses (5-25 × 103 cells/well) to the fresh assay MLC between young B6 responders (1.5 × 103 cells/well) and DBA/2 stimulators. Cultures of young B6 responders alone gave 37 960 cpm. Aged mouse shown in panel C is deficient in suppressor cell generation compared to young control whereas aged mouse in panel B is not deficient in suppressor cell generation. *Significantly different at P < 0.02 from suppressor cells generated without added supernatants.

DISCUSSION AND CONCLUSIONS Among the assays used to test the immunocompetence of old mice, the proliferative T cell response has been found to be the most adversely affected by aging and is already profoundly depressed by middle age (14--20 months) [2,3]. We have investigated here whether the defects in proliferation, and in one of the functional assays, suppression, are due to the documented reduction in IL-2 production with age [ 5 - 1 2 ] . In an indirect approach to the question, allogeneic and semi-allogeneic stimulator cells were compared in the MLC. Irradiated spleen cells (the stimulator population), although unable to divide, are still capable of responding to the presence of allogeneic cells (the responder population) with the production of lymphokines. The use of irradiated semiallogeneic cells as the stimulator population eliminates this source of IL-2. The present results show that the difference in the proliferative ability of responder cells from young and older mice is greater when fully allogeneic than when semi-allogeneic stimulator cells are used. However, the percent increase in proliferation to fully allogeneic, as opposed to semi-allogeneic, stimulators appears similar for responder cells from young and aged mice. This implies that the additional presence of lymphokines resulting from the back stimulation of the stimulator cells enhances the proliferative response of young and aged mouse spleen cells proportionally. When exogenous IL-2 rich supematant is directly added to the fully allogeneic MLC similar results are obtained. The percent increase in proliferation is similar for spleen cells

111 from young and aged mice. This increase, however, is not great enough to bring the response of the aged mice to the baseline level of the young. Increasing the amount of IL-2 rich supematant added results in a shift of the dose-response and kinetics curves of the response by the young mouse spleen cells. This is probably due to cell crowding and exhaustion of nutrients and can thus result in surprisingly low values if only single time points are studied. In previous studies [3], spleen cells from 41% of the 22 mice tested generated significantly less H-2 specific suppressor activity than did young control spleen cells in the MLC. In those experiments IL-2 rich lymphokine preparations, added with the suppressor cells to the assay cultures, were unable to overcome the suppression [3]. The suppression in this system is, therefore, not due to a competition for lymphokines by suppressor cells and fresh responder cells in the MLC. The present results show that addition of IL-2 rich supernatant to the generation cultures of suppressor cells does not affect the response of cells from young mice or from aged mice capable of control levels of suppressor T cell generation. However, the lymphokines do cause an increase in the amount of suppression generated by spleen cells from aged mice which are deficient in their suppressor T cell response. Thus, the defect in suppressor cell generation of some of the aged mice is apparently due primarily to a relative lack of lymphokine production needed for suppressor cell proliferation. One hypothesis which would account for the present results is that spleen cells from aged mice have a decreased number of precursor cells capable of responding with proliferation to allogeneic stimulators. An IL-2 source, whether from back stimulation or exogenously added, results in the same relative increase for cells from young and aged mice. It is therefore possible that the responsive cells from aged mice, are capable of the same number of division cycles in response to the lymphokines as are the cells from the young mice. Support for this hypothesis would require the use of limiting dilution assays to enumerate precursor cell frequencies. In view of the lack of information about lymphokine requirements for suppressor cell proliferation and maturation, it is difficult to decide without further experimentation whether the effect of added lymphokines is due to an increased number of precursor cells responding or to heightened proliferation of the existing precursors of suppressor cells. Apparently, the lymphokine requirement for suppressor cell generation in this MLC system is more readily satisfied than that for T cell proliferation, since addition of IL-2 rich supematants always enhanced proliferation, but only enhanced suppressor cell generation in suppressor cell deficient cultures. The present results are in disagreement with those suggesting that IL-2 supplementation can restore the proliferative response of cells from aged mice up to the level of responses of cells from young mice [6,8,10,13]. This does not negate the fact that significant improvement is seen with added IL-2 but suggests that other defects must ~lso exist. These probably lie at the level of the precursor cells;perhaps a decreased number of precursor cells or an inability of existing precursor cells to recognize allogeneic stimulators and/or to develop IL-2 receptors. In addition, as has been found for lymphocytes from elderly humans [19], cells from aged mice may be more sensitive than cells from young

112 mice to cell cycle arrest by tritrated thymidine. This would be evident as a decrease in the proliferative response. Miller [9], studying a variety of T cell functions including IL-2 production, demonstrated a decrease in precursor cell frequency with maintenance o f level o f function per responsive T cell. Findings in aged rats by Gilman et al. [5], suggested that with age there is a defect in both the production of and the ability to respond to IL-2. Joncourt et al. [12], following the response to lectins by the passage of cells through the stages o f the cell cycle, similarly concluded that, in mice, there is a decrease in both production o f and proliferative responsiveness to IL-2. Chang et al. [6] supplemented concanavalin-A stimulated cultures with exogenous lymphokine preparations on several consecutive days and achieved restoration o f the proliferative response to young control levels in this fashion. In summary, these results suggest that the defect in T cell mediated immune funtion of aged mice may be at least partially corrected by the addition o f exogenous IL-2 enriched lymphokine preparations. This is particularly applicable for T cell functions not severely affected by the aging process, i.e., suppression. REFERENCES 1 S.R.S. Gottesman and R.L. Walford, Autoimmunity and aging. In R.C. Adelman and G.S. Roth (eds.), Methods in Aging Research, Vol. XIV, CRC Press, Inc., West Palm Beach, FL, 1982, pp. 233 -279. 2 S.R.S. Gottesman, J.A. Kristie and R.L. Walford, Proliferative and cytotoxic immune functions in ageing mice. I. Sequence of decline of reactivities measured under optimal and suboptimal sensitization conditions. Immunology, 44 (1981) 607-616. 3 S.R.S. Gottesman, R.L. Walford and G.J. Thorbecke, Proliferative and cytotoxic immune functions in aging mice. II. Decreased generation of specific suppressor cells in ailoreactive cultures. Y. Immunol., 133 (1984) 1782-1787. 4 K. Smith, lnterleukin 2. Annu. Re;,. Immunol., 2 (1984) 319-333. 5 S.C. Gilman, J.S. Rosenberg and J.D. Feldman, T Lymphocytes of young and aged rats. II. Functional defects and the role of interleukin-2. J. Immunol., 128 (1982) 644-650. 6 M-P. Chang, T. Makinodan, W.J. Peterson and B.L. Strechler, Role ofT cells and adherent cells in age-related decline in routine interleukin 2 production. J. Immunol., 129 (1982) 2426-2430. 7 M.L. Thoman and W.O. Weigle, Lymphokines and aging: Interleukin-2 production and activity in aged animals. J. Immunol., 127 (1981) 2102-2106. 8 M.L. Thoman and W.O. Weigle, Cell mediated immunity in aged mice: an underlying lesion in IL-2 synthesis. Y. Immunol., 128 (1982) 2358-2361. 9 R.A. Miller, Age-associated decline in precursor frequency for different T cell-mediated reactions, with preservation of helper or cytotoxic effect per precursor c~U.Z Iramunol., 132 (1984) 63-68. 10 R.A. Miller and O. Stutman, Decline in aging mice, of theanti-2,4,6-trinitrophenyl (TNP) cytotoxic T cell response attributable to loss of Lyt-2 ", interleukin 2-producing helper cell function. Eur. J. Immunol., 11 (1981) 751-756. I I S. GiIlis,R. Kozak, M. Durante and M.E. Weksler, Immunologic~ studies of aging. Decreased production of and response to T cellgrowth factor by lymphocytes from aged humans.J. Clin.Invest,, 67 (1981) 937-942. 12 F. Joncourt, Y. Wang, F. Kristensen and A.L. De Weck, Aging and immunity: Decrease in interleukin-2 production and interleukin-2-dependent RNA-synthesis in lectin-stimulatedmurine spleen ceils.Immunobiology, .163 (1982) 521-526.

113 13 MJ~. Thoman and W.O. Weigle, Deficiency in suppressor T cell activity in aged animals. Reconstitution of this activity by interleukin-2. J. Exp. Med., 157 (1983) 2184-2189. 14 J.S. Rosenberg, S.C. Gilman and J.D. Feldman, Effects of aging on cell cooperation and lymphocyte re~ponsiveness to cytokines. J. Immunol., 130 (1983) 1754-1758. 15 S. Gillis, M.M. Ferm, W. Ou and K.A. Smith, T cell growth factor: parameters of production and a quantitative microassay for activity. J. lmmunol., 120 (1978) 2027-2032. 16 A. Peck and F. Bach, A miniaturized mouse mixed lymphocyte culture in serum free and mouse serum supplemented media. Jr. Immunol. Methods, 3 (1973) 147-164. 17 R. Hodes and W. Terry, Comparison of irradiated and mitomycin-treated mouse spleen cells as stimulating ceils in mixed leukocyte cultures and in vitro sensitization. J. Immunol., 113 (1974) 39 -44. 18 R.J. Hodes, L~[. Nadler and K.S. Hathcock, Regulatory mechanisms in cell-mediated immune responses. HI. Antigen-specific and nonspecific suppressor activities generated during MLC. J. Immunol., 119 (1977) 961-967. 19 L. Staiano-Coico, Z. Darzynkiewicz, M.R. Melamed and M.E. Weksler, Immunological studies of aging. IX. Impaired proliferation of T lymphocytes detected in elderly humans by flow cytometry. Z Immunol., 132 (1984) 1788-1792.