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Age-related changes within a suppressor T cell circuit
CELLULAR
122,20-32 ( 1989)
IMMUNOLOGY
Age-Related
Changes within a Suppressor
GINO DORIA,
CAMILLO
MANCINI,
T Cell Circuit’
AND DAMELA
FRASCA
Laboratory ofPathology, ENEA C.R.E. Casaccia, Rome, Italy Received February 61989; accepted March 28, I989 The effects of aging on cellular and molecular components of the 4-hydroxy-3-nitrophenyl acetyl-specific suppressor T (Ts) cell circuit were analyzed in vitro using inducer (Tsl), transducer (Ts~), and effector (Ts3) cells and activating factors (TsFl and TsF2) derived from young or old mice. The activation of Ts2 cells by TsFl and of Ts3 cells by TsF2 was found age-restricted, suggesting a loss of Ts2 and Ts3 cell subsets in old mice. However, the activation of Ts3 cells by small amounts of TsF2 is more efficient when both are derived from old rather than from young mice while the same level of maximum suppression is attained. Higher affinity of the interactions involved in Ts cell activation may compensate for loss of Ts cell subsets in old mice. No age restriction was found for antigen presentation to Tsl cells and for the interaction between Ts3 cells and target B cells. Thus, the effects of aging on immunosuppression result from changes within the Ts cell circuit. o 1989Academic mess, IN.
INTRODUCTION The 4-hydroxy-3nitrophenyl acetyl (NP)’ system is a well-defined experimental model of antigen-specific T cell-mediated immune suppression, characterized by the sequential interaction of various T cell subsets and soluble factors. Antigen presentation by I-A+, Jf macrophages activates an inducer T cell, Tsl (Tsi; Lyt- l+, 2-; J+; Id+), which produces an antigen-specific factor, TsFl (Tsi F; J+; Id+; nonrestricted by MHC and Igh genes; antigen-binding). In the absence of antigen, TsFl presented by I-A+, J+ macrophages activates transducer T cells, Ts2 (Tst; Lyt- l+, 2+; J+; Id-), which recognize TsFl by anti-Id receptors and are restricted by J and Igh genes. Activated Tst cells produce a factor, TsF2 (Tst F; J+; anti-Id; restricted by J and Igh genes), which, if presented by I-A+, J+ macrophages, triggers previously primed effector T cells, Ts3 (Tse; Lyt-l-, 2+; J+; Id+), to produce and release a factor, TsF3 (Tse F; J+; Id+; restricted by J and Igh genes; antigen-binding). Ts3 cells are primed by antigen presented on I-A+, J+ macrophages. TsF3 suppresses specifically responder Th cells (inhibition of proliferation and lymphokine production) and B cells (inhibition of antibody production) and arms acceptor T cells, Ts4 (Tsa; Lyt- 1-, 2+; Jf), and accep tor I-A+, J+ macrophages. Armed acceptor cells are then triggered by antigen associ’ This work was supported by ENEA-Euratom contract and by the Pasteur Institute-Cenci Bolognetti Foundation. It is publication No. 2505 of the Euratom Biology Division. * Abbreviations used: HRBC, horse red blood cells; Id, idiotype; NP, 4-hydroxy-3-nitrophenyl acetyl; NP-O-&t, NP-0-succinimide; PFC, plaque-forming cells; SC, syngeneic spleen cells; Tsl , inducer suppressor T cell; Ts2, transducer suppressor T cell; Ts3, effector suppressor T cell; TsFl, TsF2, factors produced by Tsl , Ts2 cells, respectively. 20 0008-8749189 $3.00 Copyright Q 1989 by Academic Rsr, Inc. All rights of reproduction in any form reserved.
AGING
AND SUPPRESSOR T CELLS
21
ated with J molecules to release factors that are genetically unrestricted and suppress nonspecifically responder T and B cells (1,2). Age-related alterations of the cell recognition repertoire have been suggested to occur in B (3) and T lymphocytes (4, 5). In a previous study (6) we found that the cell interactions involved in the NP-specific suppressor T cell circuit are age-restricted as in vitro activation of Ts2 cells by Ts 1 cells is less effective if these cell populations are derived from mice of a different age. Moreover, age restriction was also found to operate in the interactions between Ts2 and Ts3 cells. Thus, aging appears to induce profound changes of the recognition repertoire expressed in the Ts cell subsets of the NP-specific circuit. In the present study the analysis of the NP system has been extended using monoclonal TsF 1- and TsFZcontaining supernatants to dissect out the molecular and cellular components of the circuit for a more precise identification and quantitative evaluation of the network elements affected by senescence. MATERIALS
AND METHODS
Mice. (C57Bl/ 1OXDBA/2)F 1 male mice were produced in our animal facilities and used at the age of 3 months (young) or 18 months (old). Antigens and chemicals. Horse red blood cells (HRBC) and sheep red blood cells (SRBC) were obtained from Sclavo (Siena, Italy), and 4-hydroxy-3-nitrophenyl acetyl-O-succinimide (NP-O-Su) was purchased from Cambridge Research Biochemicals (Hariston, UK). NP-conjugated HRBC and SRBC were prepared as follows (7). Two milliliters of washed and packed RBC was suspended in 8 ml of borate-buffered saline, pH 9. NP-O-Su (20 mg in 2 ml of dimethylformamide) was added dropwise. After incubation for 10 min the reaction was stopped by addition of 40 ml of cold phosphate-buffered saline (PBS), pH 7.7, and RBC were washed three times in PBS before use. NP-conjugated syngeneic spleen cells (NP-SC) were prepared as described (7). Briefly, spleen cells from young mice (in one experiment, from old mice) sacrificed immediately after total body exposure to 2000 rad of X-rays were suspended in balanced salt solution (BSS) and adjusted to the desired cell concentration. After centrifugation, the pellet expected to contain 4 X lo8 nucleated cells was resuspended in 5 ml BSS 0. lx to lyse RBC. After 10 set 5 ml BSS 2x was added to prevent lysis of nucleated cells. After centrifugation the pellet was resuspended in 4 ml of PBS, pH 7.7, and then 100 ~1 of dimethyl sulfoxide, containing 2.4 mg of NP-O-Su, was added to the cell suspension. After 4 min 40 ml of BSS was added to stop the reaction, and, after centrifugation, NP-SC were washed twice in BSS and passed through a 100~pm nylon filter before use. Cyclophosphamide (CY) was purchased as Endoxan-Asta from Shering (Italy). Priming. Young and old mice were carrier-primed by iv injection of 2 X lo5 HRBC in 0.2 ml PBS to induce maximum numbers of helper T cells (8). After 4 days mice were sacrificed and their spleen cells were immunized in vitro with NP-HRBC. Responder cell cultures. Spleen cells from four HRBC-primed mice were suspended in complete culture medium containing RPM1 1640 (GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal calf serum (Flow Laboratories, UK), 2 mM rglutamine (GIBCO), and 10 pg/ml gentamicin (Shering, Kenilworth, NY), and cultured in 24-well tissue culture plates (No. 3524; Costar, Cambridge, MA) according to Mishell and Dutton (9). In each experiment triplicate culture wells received 0.5 ml medium containing 1 X 1O6NP-HRBC and 3 X 1O6nucleated spleen cells. Triplicate wells received no NP-HRBC and served as background controls. The anti-NP and
22
DORIA, MANCINI,
AND FRASCA
anti-HRBC antibody responses in each well were assayed on Day 4 of culture by the Cunningham and Szenberg technique (lo), with NP-SRBC or HRBC used as test antigens and evaluated as the number of direct plaque-forming cells (PFC) per culture well. For each antibody response the PFC background value, expressed by the geometric mean from triplicate wells without antigen, was subtracted from the number of PFC induced by antigen in each well. The geometric mean of net PFC numbers from triplicate wells was then calculated and reported as PFC per culture. An error factor by which the geometric mean should be multiplied or divided to obtain the variations due to one standard deviation is also given. Calculation of the geometric mean and its error factor was performed by antilog transformation of both the arithmetic mean of log PFC numbers from triplicate wells and its standard deviation. Induction of Tsl cells. NP-specific Tsl cells were activated in young and old mice by iv injection of 0.5 ml BSS containing 4 X lo7 NP-SC, prepared with SC from young mice. (In one experiment, young mice were given 4 X 105, 4 X 106, or 4 X lo7 NP-SC prepared with SC from young and old mice.) Six days later, four mice of each group were sacrificed and their spleen cells were used in vitro. TsFl-containing supernatant. This was a gift from D. H. Sherr, prepared from the B6-Ts l-29 hybridoma obtained by fusing BW 5 147 tumor cells with spleen cells from 2- to 3-month-old C57B1/6 mice (11). Subculture of Tsl and Ts2 cells. Generation of Ts2 cells in normal spleen cell populations is an antigen-independent process induced in culture by Tsl cells (12), activated in the spleens of NP-SC-injected mice, or by TsFl ( 13). Spleen cells from NPSC-injected mice and spleen cells from normal mice, hereafter designated Tsl cells and Ts2 cells, respectively, were mixed and subcultured in the same wells of Costar No. 3524 tissue plates under optimal conditions previously established for spleen cells from young and old mice (6). Mixing of Ts 1 cells with Ts2 cells was performed by combining spleen cells from mice of the same age (young or old). Unmixed Ts2 cells served as control. Each cell suspension was prepared from a pool of four spleens and adjusted to a concentration of 8 X lo6 nucleated cells/ml complete medium. Equal volumes of the Ts 1 and Ts2 cell suspensions were mixed so that 1 ml of the final suspension contained 4 X 1O6Ts 1 cells and 4 X 1O6Ts2 cells. Each final suspension of unmixed or mixed spleen cells was distributed in 4-24 wells (0.5 ml/well) and cells were cultured for 4 days. In some experiments, 0.5 ml of the unmixed Ts2 cell suspension from young or old mice was cultured with a monoclonal TsFl -containing supernatant which was added daily, in variable amounts (10 or 40 pl), to the Ts2 cell culture wells for 4 days. The addition of complete medium as control was used in preliminary experiments but was then abandoned because it had no effect. Thereafter, in one series of experiments cells harvested from the wells of each group were pooled, washed, centrifuged, resuspended in complete medium, and adjusted to a final concentration of 1.5 X lo7 nucleated cells/ml. Samples (0.1 ml) of each cell suspension were then added to responder cell culture wells 1 day before the PFC assays. In another series of experiments cell suspensions from the wells of each group were pooled and centrifuged, and their TsF2-containing supematant was collected and used immediately or frozen at -20°C. When used, graded aliquots of each supernatant were adjusted to a final volume, by adding complementary aliquots of complete medium, and then a constant aliquot, usually 300-500 ~1, of the final solutions containing different amounts of the original supematant was added to responder cell culture wells 1 day before the PFC assays.
AGING
AND SUPPRESSOR T CELLS
23
Ts3 cell depletion and enrichment. Depletion of Ts3 cell precursors was induced in young mice by CY treatment (13), as follows. One day after HRBC priming, mice were injected ip with 20 mg/kg CY in 0.2-0.25 ml PBS and, 3 days later, their spleen cells were used as responder cells in culture with NP-HRBC. Ts3 cell enrichment was performed by negative selection on plastic plates (Falcon, 3003 F) coated overnight with 5 ml of affinity-purified goat anti-mouse Ig antibody (100 &ml) and repeatedly washed with PBS before use (14). Briefly, responder spleen cells from HRBC-primed young or old mice were cultured with NP-HRBC for 3 days and then harvested, pooled, and transferred onto antibody-coated plates. Each plate received 5 X 10’ cells in 10 ml of complete medium without fetal calf serum and was incubated at 4°C for 1 hr. Nonadherent cells were recovered by gentle swirling and were usually more than 95% Thy-l .2+ and less than 5% Ig+, as detected by indirect fluorescence. This T cellenriched population was assumed to be enriched in Ts3 cells. Percentage of suppression of PFC responses. Suppression of anti-NP and antiHRBC PFC responses was evaluated by the geometric means of net PFC numbers from triplicate cultures that received either subcultured cells or their supernatants, as follows: Percentage suppression = 1 -
PFC (Tsl + Ts2) x loo PFC (Ts2) .
Ts 1 stands for Tsl cells or TsFl in subculture; Ts2 stands for Ts2 cells in subculture. An error factor, by which the percentage should be multiplied or divided to obtain the variations due to one standard error, was calculated by the antilog transformation of the standard error obtained by the application of Fieller’s theorem ( 15) to log values of geometric means and of their error factors. Estimates from independent experiments were combined by using log values of each percentage (Pi) and of its error factor (EFJ as follows. Each log Pi was multiplied by l/(log EFi)*, and the sum of log Pi/(lOg EFJ* was divided by the sum of l/(log EFJ* to provide the weighted mean of log P. The antilog transformation gives the weighted geometric mean of P. An error factor of the weighted mean was also calculated according to Cochran ( 16). Comparison among suppression percentages was performed by the t test using log values of the percentages and of their error factors. All data were computed as previously described ( 17). RESULTS Suppression by subcultured cells. Activation of Ts2 cells in subculture was induced by a monoclonal TsFl -containing supernatant that was obtained from a hybridoma originally derived from spleen cells of young mice. TsF 1 was used to replace Ts 1 cells and 10 or 40 ~1 of the supernatant was added daily to Ts2 cells in subculture for 4 days. Thereafter, subcultured cells were pooled and washed, and 1.5 X 1O6cells were added to 3-day cultures of responder cells from young mice. Each culture well was assayed on Day 4 for anti-NP and anti-HRBC PFC responses. Results in Table 1 demonstrate that if sufficient amounts of TsFl are added to subcultures, Ts2 cells from young mice but not Ts2 cells from old mice can be activated to transduce NPspecific suppression. Control subcultures indicate that the interaction between Tsl cells and Ts2 cells from old mice is very efficient, although to a lesser extent than the
24
DORIA, MANCINI,
AND FRASCA
TABLE 1 Age-Restricted Activation of Ts2 Cells That Transduce NP-Specific Suppression of Responder Cells from Young Mice Subculture EXPT. 1
2
3
Tsl cells
TsFl
Ts2 cells
Anti-NP PFC/culture
Young Old -
+ +
Young Young Young Old Old Old
1883(1.05) 2098 (1.02) 896(1.25) 1948(1.07) 1996(1.07) 1496 (1.07) 1956(1.12)
Young Old -
+ +
Young Young Young Old Old Old
3831 (1.27) 4244 ( 1.20) 1866(1.24) 2973 (1.35) 4598(1.18) 3084 (1.20) 4982(1.14)
Young Old -
+ +
Young Young Young Old Old Old
4102 (1.09) 5068 (1.20) 267 1 (1.05) 3315 (1.11) 3988 (1.12) 2827 (1.09) 3860(1.11)
w Suppression
51(1.45) 7 (1.05) 25 (1.06) 2(1.08)
56(1.17) 30(1.23) 33(1.15) 0
47(1.11) 35(1.13) 29 ( 1.09) 3(1.09)
Anti-HRBC PFC/culture 4501(1.09) 4911 (1.09) 4948(1.05) 4999 ( 1.05) 4840 (1.05) 4746(1.11) 4787 (1.05) 7527 (1.03) 8587 (1.05) 8081(1.05) 8109(1.05) 7817(1.03) 8181(1.12) 8360 (1.09) 1781 (1.05) 1772(1.07) 1747(1.05) 1790(1.09) 1751(1.09) 1832(1.09) 1727 (1.05)
% Suppression
0 0 2 (1.07) l(l.05)
6(1.05) 6(1.04) 0 0
l(l.06) 0 0 l(l.06)
Note. Spleen cells from young or old mice were subcultured in 0.5 ml of complete medium, as follows: 4 X 1O6Ts2 cells alone or with TsFl ; 2 X 106 Tsl cells and 2 X 1O6Ts2 cells. TsFl was added to subcultures daily, four times from Day 0 to Day 3: 10 pi/day in Experiment 1 or 40 pi/day in Experiments 2 and 3. On Day 4, subcultured cells were harvested, pooled, and washed, and 1.5 X lo6 cells were transferred to Day 3 responder cell cultures each of which was assayed for anti-NP and anti-HRBC PFC responses on the subsequent day. Anti-NP PFC/culture background responses without antigen: 5 14 (Experiment 1), I8 13 (Experiment 2), and 3 192 (Experiment 3). Anti-HRBC PFC/culture background responses without antigen: 220 (Experiment l), 176 (Experiment 2), and 110 (Experiment 3). Each background value (geometric mean from triplicate cultures) has been subtracted from the number of PFC induced by antigen in individual cultures. Geometric means of net PFC numbers from triplicate cultures are reported in the table. Numbers in parentheses are error factors by which means and percentages should be multiplied or divided to obtain variations due to one standard deviation and one standard error, respectively.
interaction between Ts 1 cells and Ts2 cells from young mice, confuming our previous findings (6). Suppression by subculture supernatant. A major advantage of using subculture supernatant rather than subcultured cells to transduce suppression is the possibility of adding increasing amounts of TsF2 to responder cells to attain maximum levels of suppression avoiding exceedingly high cell density in responder cell cultures. In this series of experiments 2 X lo6 Tsl cells and 2 X lo6 Ts2 cells from mice of the same age (young or old) were mixed in 0.5 ml/well and subcultured for 4 days. Unmixed Ts2 cells (4 X 106/0.5 ml) served as control. Thereafter, subculture supernatants were collected and added to responder cell cultures.
AGING
25
AND SUPPRESSOR T CELLS TABLE 2
Requirement of Cell Interaction for Generation of Subculture Supematant That Transduces Suppression of Responder Cells from Young Mice Cells in subculture Subcultured cells from mice Young
Ts 1 Tsl and Ts2
Subculture supematant (~1) added to responder cells
Anti-NP PFC per culture
% Suppression
-
2600(1.05) 2482 (1.03) 1055 (1.05) 1921 (1.05)
58 (1.04) 23 (1.03)
2349(1.02)
5 (1.03)
2336 (1.02) 1667 (1.05) 2149(1.02)
29(1.03) 8 (1.01)
2362 (1.02)
0
-
-
+
+
+ -
-
300 300 300
+
-+ -
-+ + -
150 150 I 300 300 300
+
--
-+
150 150 I
+ Old
Ts2
Note. Spleen cells from young or old mice were subcultured in 0.5 ml of complete medium, as follows: 4 X lo6 Tsl cells alone; 2 X lo6 Tsl cells and 2 X IO6 Ts2 cells; 4 X lo6 Ts2 cells alone. On Day 4, 300 ~1 of subculture supematant were transferred to Day 3 responder cell cultures which were assayed for antiNP PFC responses on the subsequent day. Anti-NP PFC/culture background response: 6 IO. The background value (geometric mean from triplicate cultures) has been subtracted from the number of PFC induced by antigen in individual cultures. Geometric means of net PFC numbers from triplicate cultures are reported in the table. Numbers in parentheses are error factors by which means and percentages should be multiplied or divided to obtain variations due to one standard deviation and one standard error, respectively.
Data from a preliminary experiment (Table 2) indicate that the supematant is very effective in transducing suppression if it is derived from a mixture of subcultured Tsl and Ts2 cells. Supematants from subcultures of unmixed cells have negligible effects, except for supematant from subcultured Tsl cells as expected from the fact that these spleen cells from NP-SC-injected mice comprise activated Tsl cells and Ts2 cells in unknown proportion. The data also show that the supematant activity depends on cell interaction in subculture because the pool of equal volumes of supematants from unmixed Ts 1 and Ts2 cells is devoid of sizable effects. It should be noted that supematants from subcultured cells from young mice are more effective than supematants derived from old mice when added to responder cells from young mice, suggesting that the interaction between TsF2 and Ts3 cells is age-restricted. The Tsl cell transfer from mice injected with NP-SC may convey NP-specific antibody-forming cells and antigen to subculture. However, the possibility that the supernatant activity is mediated by anti-NP antibodies produced in subculture is ruled out by the finding that comparable numbers of anti-NP PFC/spleen were found in young mice, either normal (360 PFC) or injected with 4 X 10’ NP-SC six days before sacrifice (420 PFC). Moreover, the same anti-NP antibody response was also found in 4day subcultures of 4 X lo6 mixed Tsl and Ts2 cells (890 PFC) or 4 X lo6 unmixed Ts2 cells (870 PFC) from young mice. In one experiment, 4 X 105, 4 X 106, or 4 X 10’ NP-SC, prepared with SC from young and old mice, were injected in young mice to induce Ts 1 cells. Mixed Ts 1 cells and Ts2 cells or unmixed Ts2 cells from young mice were subcultured for 4 days and
26
DORIA, MANCINI,
AND FXASCA
60
40
20
0 Subculture
supernatant
added
to
responder
cells
FIG. 1. Percent suppression of responder cells from young mice transduced by supernatants from subcultures containing Tsl and Ts2 cells from young mice. Tsl cells were activated by injection of 4 X lo5 (a q ), 4 X lo6 (A, A), or 4 X 10’ (0,O) NP-SC. Solid symbols refer to young SC donors, and open symbols refer to old SC donors. Error factors range from 1.02 to 1.06.
then their supernatants were transferred to responder cells from young mice 1 day before the anti-NP PFC assay. Figure 1 shows that suppression increases with the amount of the added supernatant up to a maximum level, reflecting the number of injected NP-SC and, therefore, the number of activated Ts 1 cells. No difference was found between the two NP-SC preparations, from young and old mice, suggesting that Ts 1 cell activation in young mice is not age-restricted. Age-related changes in the interaction between TsF2 and Ts3 cells were investigated as follows. In each experiment, Ts 1 cells and Ts2 cells from young or old mice were subcultured for 4 days and then graded amounts of the subculture supematants were added to responder cells from young or old mice. Using responder cells from young mice in two experiments and from old mice in four experiments it was found that after the addition of increasing amounts of supematant from subcultures containing Ts 1 cells and Ts2 cells from young or old mice the percentage suppression of the anti-NP PFC response increases up to plateau levels in all experiments (Fig. 2). However, using responder cells from young mice, suppression reaches a higher plateau level when the supematant is derived from young mice than when it is derived from old mice. Conversely, using responder cells from old mice, the two supematants bring about approximately the same plateau of suppression but the supematant derived from old mice is much more efficient than that derived from young mice. Hence, while 50 ~1 of the former supematant is sufficient to transduce maximum suppression, 300 ~1 of the latter is needed to reach the plateau level. The suppression profiles depicted in Fig. 2 demonstrate that the TsF2-mediated transduction of suppression of the anti-NP PFC response is age-restricted. The antigen specificity of suppression was assessed using responder cells from young or old mice and subculture supematant from young or old mice in optimal amounts to transduce maximum suppression. Results in Table 3 establish that sup pression is NP-specific and confirm the age restriction described in Fig. 2. Table 4 summarizes the results from four to seven experiments (pooled data from Fig. 2 and Table 3) showing not only that transduction of suppression is age-restricted but also that the same level of maximum suppression may be achieved under age-
AGING
27
AND SUPPRESSOR T CELLS
60 -
Responder
'
[
cells
F
100 Subculture
200 supernatant
300 added
fro+n
young
from
ild
mice
mice
400 to
responder
1
500
ul
cells
FIG. 2. Percent suppression of responder cells from young or old mice transduced by supematants from subcultures containing Tsl and Ts2 cells from young (0) or old (0) mice. Symbols represent weighted means from two to four experiments. Error factors range from 1.02 to 1.11.
matched conditions. Thus, the supematant-responder cell mixture yields equal levels of maximum suppression in the young-young and old-old combinations. The evidence so far presented for age-restricted transduction of suppression implies that TsF2 and Ts3 cells may be derived from mice of the same age but may also require age matching of responder B cells. This issue was investigated by use of TsF2containing supematant derived from old mice to transduce suppression of responder cells from young mice, after depletion of Ts3 cell precursors by in vivo CY treatment, and in vitro addition of activated Ts3 cells from old mice. Table 5 presents all data from one experiment. The efficiency of the CY treatment to deplete responder spleen cells of Ts3 cell precursors is shown by the difference in PFC response between groups I and VI as well as between the background control groups and by the negligible level of suppression in groups VIII and X. The efficiency of adding activated Ts3 cells from young or old mice to reconstitute responder cell populations sensitive to subculture supematants is demonstrated by the same loss (50%) of suppression from groups III (58%) to V (30%) as that from groups XII (47%) to XIV (24%) and by the comparable levels of suppression observed in groups XII (48%) and XVI (46%) as expected from data in Fig. 2. The critical result ofthis experiment, however, is the difference in percentage suppression between groups XIV (24%) and XVIII (52%). Althougb B cells in group XVIII are from young mice, the replacement of Ts3 cells from young mice with Ts3 cells from old mice increases the sensitivity of the responder cells to the subculture supematant derived from old mice up to the same level exhibited by responder cells from old mice (Fig. 2 and Table 4). It appears, therefore, that age matching between TsF2 and Ts3 cells is necessary and sufficient for effective suppression of responder cells. The experiment was repeated and provided similar results. DISCUSSION This work extends our previous investigation on the effects of aging on immunosuppression (6) and indicates the NP system as a reliable and suitable experimental
28
DORIA, MANCINI,
AND FRASCA
TABLE 3 NP Specificity and Age Restriction of Maximum Suppression Transduced by Subculture Supematant
EXPT 1
2
3
4
5a
5b
Responder cells from mice Young
Young
Young
Young
Young
Old
Subculture Ts 1 cells
-
Ts2 cells
-
Anti-NP PFcfculture
Young Old
Young Young Old Old
4176 (1.07) 4173 (1.09) 2245 (1.16) 4189(1.12) 2697 (1.16)
Young Old
Young Young Old Old
4860(1.20) 4762 (1.22) 1960(1.14) 4799(1.14) 2579 (1.09)
Young Old
Young Young Old Old
7418(1.05) 7262(1.05) 3556 (1.05) 7615 (1.02) 4465 (1.03)
Young Old
Young Young Old Old
3296 (1.09) 3166(1.09) 1508 (1.16) 3278 (1.03) 1967(1.11)
Young Old
Young Young Old Old
2312(1.07) 2324 (1.07) 1079(1.18) 2328 (1.03) 1231(1.22)
Young Old
Young Young Old Old
505 (1.12) 498 (1.27) 304 ( 1.20) 520(1.07) 263(1.24)
96 Suppression
Anti-HRBC PFC/culture
% Suppression
46(1.11)
2366(1.02) 2337(1.07) 2379(1.03) 2312(1.03) 2336 ( 1.07)
0
4070 (1.03) 3895 (1.05) 4004(1.03) 3947 (1.05) 3951(1.02)
0
36(1.11)
59(1.15) 46(1.10)
51(1.05) 41(1.02)
52(1.10) 40(1.06)
54(1.11) 47 (1.41)
39 (1.20) 49(1.14)
0
0
1228 (1.05) 1246(1.09) 1229(1.02) 1255 (1.03) 1260(1.07)
l(l.05)
1388(1.05) 1368 (1.05) 1309 (1.03) 1299 (1.05) 1282 (1.64)
4(1.03)
1681 (1.02) 1687 (1.03) 1708(1.12) 1868 (1.14) 1727 (1.03)
0
525 (1.09) SOS(l.16) 585 (1.09) 565(1.11) 612 (1.09)
0
l(l.04)
8 (1.09)
0 0
Note. Spleen cells from young or old mice were subcultured in 0.5 ml of complete medium, as follows: 2 X lo6 Tsl cells and 2 X lo6 Ts2 cells, or 4 X lo6 Ts2 cells alone. On Day 4, subculture supematants were collected and transferred to Day 3 responder cell cultures. Cultures in all groups received 300 ~1 of supematant except in the last two groups of Experiment 5b in which responder cell cultures received 50 ~1 of supematant. Each responder cell culture was assayed for anti-NP and anti-HRBC PFC responses on Day 4. Anti-NP PFC/culture background responses without antigen: 776 (Experiment l), 639 (Experiment 2), 1318 (Experiment 3), 799 (Experiment 4), 457 (Experiment 5a), and 225 (Experiment 5b). Anti-HRBC PFC/culture background responseswithout antigen: 366 (Experiment 1), 101 (Experiment 2), 197 (Experiment 3), 130 (Experiment 4), 78 (Experiment 5a), and 66 (Experiment 5b). Each background value (geometric mean from triplicate cultures) has been subtracted from the number of PFC induced by antigen in individual cultures. Geometric means of net PFC numbers from triplicate cultures are reported in the table. Numbers in parentheses are error factors by which means and percentages should be multiplied or divided to obtain variations due to one standard deviation and one standard error, respectively.
AGING
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AND SUPPRESSOR T CELLS TABLE 4
Maximum Suppression of Responder Cells Transduced by Subculture Supematant 96 Suppression of responder cells from mice Subcultured cells from mice
yaw!
Old
Young
Old
P
Note. Percentages are weighted means from 4 to 7 experiments. Error factors range from 1.02 to 1.06. NS stands for not significant at P = 0.60.
model for quantitative studies of the molecular and cellular elements involved in the induction, transduction, and effector phase of antigen-specific suppression. The number of activated Tsl cells depends on the NP-SC dose as larger doses of injected NP-SC yield subculture supernatants with higher efficiency in transducing suppression when added in graded amounts to responder cell cultures (Fig. 1). Since a plateau level of suppression was attained for each NP-SC dose, this finding suggests the existence of Ts 1 cell subsets with different thresholds of activation, each of which is recognized by a specific Ts2 subset that, in turn, triggers a complementary Ts3 cell subset. Different Id and anti-Id receptors may be the recognition elements of the different cell subsets. The data also show that NP-SC derived from young or old mice induce the same levels of suppression of responder cells from young mice. Thus, whatever the mechanism of antigen presentation to Ts cells in the induction phase ( 1, 1g), activation of Ts 1 cells is not age-restricted. Activation of Ts2 cells by Ts 1 cells is mediated by Id+ TsFl recognized by anti-Id Ts2 cell receptors ( 1, 2). Activation of Ts2 cells from young, but not from old, mice is induced in subculture by a monoclonal TsFl-containing supematant obtained from a hybridoma originally derived from spleen cells of young mice (Table 1). This age restriction is in keeping with our previous finding of a poor interaction in subculture between Tsl cells from young mice and Ts2 cells from old mice (6) and suggests that age mismatching negatively affects the idiotypic recognition involved in Ts2 cell activation. The data are compatible with two possibilities. (i) The idiotypic repertoire undergoes qualitative changes with aging and therefore the Id+ TsFl produced by Tsl cells from young mice is not recognized by anti-Id Ts2 cells from old mice. (ii) If there are subsets of Tsl cells and Ts2 cells characterized by different Id and anti-Id receptors, the loss of a Ts2 cell subset in old mice may completely impair the recognition ofthe monoclonal TsFl produced by a Tsl cell subset from young mice. Possibility (ii) is favored by the results in Table 1 and Fig. 1. The interaction between TsF2-containing subculture supematant and the Ts3 cells activated by antigen in responder cell culture is age-restricted (Fig. 2). Suppression of responder cells from young mice attains a higher plateau level when the subculture supematant is derived from young rather than from old mice. Conversely, suppres-
30
DORIA, MANCINI,
AND FRASCA
TABLE 5 Age-Related Changes in the Interaction between Subculture Supematant and Ts3 Cells Subculture Group
Tsl cells
I II III IV V VI VII VIII IX X XI XII XIII XIV xv XVI XVII XVIII
Young Old Young Old Young Old Young Old
Responder cells from young mice
Ts2 cells
CY treatment
Young Young Old Old Young Young Old Old Young Young Old Old Young Young Old Old
+ + + + + + + + + + + + +
Added Ts3 cells
Young Young Young Young Old Old Old Old
Anti-NP PFC/culture 1015 (1.03) 981(1.05) 408 (1.03) 955 (1.02) 668 (1.05) 1267 (1.03) 1214(1.02) 1129(1.05) 1267(1.02) 1186(1.05) 1267(1.02) 665 (1.09) 1240 ( 1.03) 937(1.11) 1265 (1.05) 681 (1.18) 1220(1.02) 591(1.12)
% Suppression
58 (1.04) 30(1.03) 7 (1.03) 6(1.03) 47 (1.05) 24(1.07) 46(1.11) 52 (1.07)
Note. Spleen cells from young or old mice were subcultured in 0.5 ml of complete medium, as follows: 2 X lo6 Tsl cells and 2 X lo6 Ts2 cells, or 4 X IO6 Ts2 cells alone. On Day 4, subculture supematants were collected and 300 pl was transferred to responder cell cultures, except groups I and VI, 1 day before the anti-NP PFC assay.Responder spleen cells (3 X 106) from HRBC-primed and CY-treated (+) or untreated (-) young mice were cultured with NP-HRBC for 4 days and then assayed for the anti-NP PFC response. On Day 3, cultures of Ts3 cell precursor-depleted responder cells from CY-treated mice received no further cells (groups VI-X) or 2 X 10’ cells, after enrichment in T cells including NP-activated Ts3 cells, from 3 day cultures containing NP-HRBC and responder cells from HRBC-primed young (groups XI-XIV) or old (groups XV-XVIII) mice. Anti-NP PFC/culture background responses without antigen: 271 for cells from CY-untreated mice and 339 for cells from CY-treated mice. Each background value (geometric mean from triplicate cultures) has been subtracted from the number of PFC induced by antigen in individual cultures of cells from CY-untreated or CY-treated mice. Geometric means of net PFC numbers from triplicate cultures are reported in the table. Numbers in parentheses are error factors by which means and percentages should be multiplied or divided to obtain variations due to one standard deviation and one standard error, respectively.
sion of responder cells from old mice reaches approximately the same plateau level when the subculture supernatant is derived from young or old mice, but the supematant from old mice is much more efficient than that from young mice when added in small amounts. If the maximum levels of suppression are compared (Table 4) transduction of suppression appears to be equally effective when age-matched TsF2 and Ts3 cells are used, both from young and old mice. These data are compatible with the existence of different anti-Id TsF2 subsets which specifically recognize complementary Id+ Ts3 cell subsets. The loss of one or more TsF2 and Ts3 cell subsets in old mice may explain why the subculture supematant derived from old mice is less efficient than the supematant from young mice to transduce maximum suppression of responder cells from young mice and why the supematant from old mice is at least as effective as the supernatant from young mice to maximally stimulate Ts3
AGING
31
AND SUPPRESSOR T CELLS
Ts3
Ts2
Tsl (LYt-1)
(Lyt-12)
TsFl
w
(Lyt-2)
TsF2
.
FIG. 3. A minimal model for age-related changes in Ts cell subsets of inducer (Tsl), transducer (Ts~), and effector (Ts3) cell populations of the NP-specific suppressor circuit. 0 and n represent idiotypes associated with anti-NP receptors.
cells from old mice. The high efficiency of the small amounts of subculture supernatant from old mice to transduce suppression of responder cells from old mice suggests that affinity of the anti-Id TsF2 and Id+ Ts3 cell reaction increases with aging. This feature may also explain why small amounts of subculture supernatants from young or old mice are equally effective in transducing suppression of responder cells from young mice although old mice have lost Ts2 and Ts3 cell subsets. The possibility of an age-related increase in Ts cell Id anti-Id binding affinity need not be surprising since high-affinity B cell clones do not disappear with aging, but are simply masked by auto-anti-Id (high affinity) antibodies ( 19). Age matching between TsF2 and Ts3 cells is necessary and sufficient for effective suppression of responder cells (Table 5), suggesting that mismatching between Ts3 and B cells is irrelevant. Thus, age restriction is operating within a suppressor T cell circuit, without affecting antigen presentation to Tsl cells or the interaction between Ts3 cells and target B cells. The model proposed in Fig. 3 offers an interpretation for the changes in antigenspecific immunosuppression observed in aging mice. The data are more easily explained by subset deletions in Ts 1, Ts2, and Ts3 cell populations rather than by qualitative modifications of the Ts idiotypic repertoire. The latter possibility might result from intrinsic accumulation of mutations or epigenetic errors (20) or be secondary to changes in the B cell Id anti-Id repertoire (2 l), some of which are known to occur in aging (3). The loss of Ts cell subsets may be a consequence of thymus involution which has been shown to decrease several T cell functions (22). However, studies on Ts cells have yielded controversial results as immunosuppression appears to increase (23-24), decrease (3 1, 32, 34-39), or remain unchanged (29) in aging mice and humans. This controversy reflects the large variety of experimental models and methods used which may deal with nonidentical parts of the same or different Ts cell circuits and, therefore, are likely to provide dissimilar results. The present analysis of the NPspecific Ts cell circuit suggests that aging affects immunosuppression by loss of Ts cell subsets. Yet, under age-matched conditions, transduction of suppression is more effective when subculture supematant and Ts3 cells are derived from old rather than
32
DORIA, MANCINI,
AND FRASCA
from young mice while the same level of maximum suppression is attained. This observation is consistent with the possibility that higher affinity of the interactions involved in Ts cell activation compensates to some extent for the loss of Ts cell subsets in old mice. Furthermore, it is conceivable that during senescence a greater inducibility of Tsl cells may help to counteract the effects of a decrease in Ts cell subsets that results from thymus involution. This possibility, previously shown to occur for the azobenzenearsonate system (33), is presently under investigation for the NP system in our laboratory. REFERENCES 1. Dorf, M. E., and Benacerraf, B., Annu. Rev. Immunol. 2,127, 1984. 2. Asherson, G. L., Colizzi, V., and Zembala, M., Annu. Rev. Immunol. 4,37, 1986. 3. Goidl, E. A., Thorbecke, G. J., Weksler, M. E., and !&kind, G. W., Proc. Natl. Acad. Sci. USA 77, 6788,198O.
Gorczynski, R. M., Kennedy, M., and McRae, S., Cell. Immunol. 75,226, 1983. 5. Hausman, P. B., Goidl, E. A., Siskind, G. W., and Weksler, M. E., J. Immunol. 134,3802, 1985. 6. Doria, G., Mancini, C., Frasca, D., and Adorini, L., J. Immunol. 139, 1419, 1987. 7. Sherr, D. H., Ju, S.-T., Weinberger, J. Z., Benacerraf, B., and Dorf, M. E., J. Exp. Med. 153, 640,
4.
1981. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
27. 28. 29. 30. 31. 32.
33. 34. 35. 36. 37. 38. 39.
Doria, G., D’Agostaro, G., and Garavini, M., Cell. Immunol. 53, 195, 1980. Mishell, R. I., and Dutton, R. W., J. Exp. Med. 126,423, 1967. Cunningham, A. J., and Szenberg, A., Immunology 14,599,1968. Okuda, K., Minami, M., Sherr, D. H., and Dorf M. E., J. Exp. Med. 154,468,198 1. Sherr, D. H., Vietor, H. E., Liu, Y. N., and Dorf, M. E., J. Immunol. 133,2417, 1984. Hausman, P. B., Sherr, D. H., and Dorf, M. E., J. Immunol. 134, 1388,1985. Mage, M. G., McHugh, L. L., and Rothstein, T. L., J. Immunol. Methods 15,47, 1977. Finney, D. J., “Statistical Method in Biological Assay.” Griffin, London, 1978. Cochran, W. G., Biometrics 10, 101, 1954. Sette, A., Adorini, L., Mancini, C., Marubini, E., and Doria, G., J. Immunol. Methods 112,91, 1988. Jenkins, M. K., Pardoll, D. M., Mizuguchi, J., Quill, H., and Schwartz, R. H., Immunol. Rev. 95, 113, 1987. McEvoy, S. J. M., and Goidl, E. A., Aging Immunol. Infect. Dis. 1,47, 1988. Holliday, R., Science 238, 163, 1987. Sy, M.-S., and Benacetraf, B., Immunol. Rev. 101, 133, 1988. Doria, G., Adorini, L., and Frasca, D., In “Aging and the Immune Response. Cellular and Humoral Aspects” (E. A. Goidl, Ed.), p. 143. Dekker, New York, 1987. Gerbase-De Lima, M., Meredith, P., and Walford, R. L., Fed. Proc. 34, 159, 1975. Goidl, E. A., Innes, J. B., and Weksler, M. E., J. Exp. Med. 144, 1037, 1976. Segre, D., and Segre, M., J. Immunol. 116,735, 1976. Makinodan, T., Albright, J. W., Good, P. I., Peter, C. P., and Heidrick, M. L., Immunology 31,903, 1976. Roder, J. C., Duwe, A. K., Bell, D. A., and Singhal, S. K., Immunology 35,837, 1978. De Kruyff, R. H., Kim, Y. T., Siskind, G. W., and Weksler, M. E., J. Immunol. 125, 142, 1980. Callard, R. E., de St. Groth, B. F., Basten, A., and McKenzie, I. F. C., J. Immunol. 124,52, 1980. Liu, J. J., Segre, M., and Segre, D., Cell. Immunol. 66,372, 1982. Amagai, T., Nakano, K., and Cinader, B., &and. J. Immunol. 16,217, 1982. Globerson, A., Abel, L., Barzilay, M., and Zan-Bar, I., Mech. Ageing Dev. 19,293, 1982. Doria, G., Mancini, C., and Adorini, L., Proc. Natl. Acad. Sci. USA 79,3803, 1982. Gottesman, S. R. S., Edington, J. M., and Thorbecke, G. J., J. Immunol. 140, 1783, 1988. Barthoid, D. R., Kysela, S., and Steinberg, A. D., J. Immunol. 122,9, 1974. Krakauer, R. S., Waldmann, T. A., and Strober, W., .J.Exp. Med. 144,662, 1976. Halgren, H., and Yunis, E., J. Immunol. 118,2004, 1977. Thoman, M. L., and Weigle, W. O., J. Exp. Med. 157,2184,1983. Gottesman, S. R. S., Walford, R. L., and Thorbecke, G. J., J. Immunol. 133, 1782, 1984.
122,20-32 ( 1989)
IMMUNOLOGY
Age-Related
Changes within a Suppressor
GINO DORIA,
CAMILLO
MANCINI,
T Cell Circuit’
AND DAMELA
FRASCA
Laboratory ofPathology, ENEA C.R.E. Casaccia, Rome, Italy Received February 61989; accepted March 28, I989 The effects of aging on cellular and molecular components of the 4-hydroxy-3-nitrophenyl acetyl-specific suppressor T (Ts) cell circuit were analyzed in vitro using inducer (Tsl), transducer (Ts~), and effector (Ts3) cells and activating factors (TsFl and TsF2) derived from young or old mice. The activation of Ts2 cells by TsFl and of Ts3 cells by TsF2 was found age-restricted, suggesting a loss of Ts2 and Ts3 cell subsets in old mice. However, the activation of Ts3 cells by small amounts of TsF2 is more efficient when both are derived from old rather than from young mice while the same level of maximum suppression is attained. Higher affinity of the interactions involved in Ts cell activation may compensate for loss of Ts cell subsets in old mice. No age restriction was found for antigen presentation to Tsl cells and for the interaction between Ts3 cells and target B cells. Thus, the effects of aging on immunosuppression result from changes within the Ts cell circuit. o 1989Academic mess, IN.
INTRODUCTION The 4-hydroxy-3nitrophenyl acetyl (NP)’ system is a well-defined experimental model of antigen-specific T cell-mediated immune suppression, characterized by the sequential interaction of various T cell subsets and soluble factors. Antigen presentation by I-A+, Jf macrophages activates an inducer T cell, Tsl (Tsi; Lyt- l+, 2-; J+; Id+), which produces an antigen-specific factor, TsFl (Tsi F; J+; Id+; nonrestricted by MHC and Igh genes; antigen-binding). In the absence of antigen, TsFl presented by I-A+, J+ macrophages activates transducer T cells, Ts2 (Tst; Lyt- l+, 2+; J+; Id-), which recognize TsFl by anti-Id receptors and are restricted by J and Igh genes. Activated Tst cells produce a factor, TsF2 (Tst F; J+; anti-Id; restricted by J and Igh genes), which, if presented by I-A+, J+ macrophages, triggers previously primed effector T cells, Ts3 (Tse; Lyt-l-, 2+; J+; Id+), to produce and release a factor, TsF3 (Tse F; J+; Id+; restricted by J and Igh genes; antigen-binding). Ts3 cells are primed by antigen presented on I-A+, J+ macrophages. TsF3 suppresses specifically responder Th cells (inhibition of proliferation and lymphokine production) and B cells (inhibition of antibody production) and arms acceptor T cells, Ts4 (Tsa; Lyt- 1-, 2+; Jf), and accep tor I-A+, J+ macrophages. Armed acceptor cells are then triggered by antigen associ’ This work was supported by ENEA-Euratom contract and by the Pasteur Institute-Cenci Bolognetti Foundation. It is publication No. 2505 of the Euratom Biology Division. * Abbreviations used: HRBC, horse red blood cells; Id, idiotype; NP, 4-hydroxy-3-nitrophenyl acetyl; NP-O-&t, NP-0-succinimide; PFC, plaque-forming cells; SC, syngeneic spleen cells; Tsl , inducer suppressor T cell; Ts2, transducer suppressor T cell; Ts3, effector suppressor T cell; TsFl, TsF2, factors produced by Tsl , Ts2 cells, respectively. 20 0008-8749189 $3.00 Copyright Q 1989 by Academic Rsr, Inc. All rights of reproduction in any form reserved.
AGING
AND SUPPRESSOR T CELLS
21
ated with J molecules to release factors that are genetically unrestricted and suppress nonspecifically responder T and B cells (1,2). Age-related alterations of the cell recognition repertoire have been suggested to occur in B (3) and T lymphocytes (4, 5). In a previous study (6) we found that the cell interactions involved in the NP-specific suppressor T cell circuit are age-restricted as in vitro activation of Ts2 cells by Ts 1 cells is less effective if these cell populations are derived from mice of a different age. Moreover, age restriction was also found to operate in the interactions between Ts2 and Ts3 cells. Thus, aging appears to induce profound changes of the recognition repertoire expressed in the Ts cell subsets of the NP-specific circuit. In the present study the analysis of the NP system has been extended using monoclonal TsF 1- and TsFZcontaining supernatants to dissect out the molecular and cellular components of the circuit for a more precise identification and quantitative evaluation of the network elements affected by senescence. MATERIALS
AND METHODS
Mice. (C57Bl/ 1OXDBA/2)F 1 male mice were produced in our animal facilities and used at the age of 3 months (young) or 18 months (old). Antigens and chemicals. Horse red blood cells (HRBC) and sheep red blood cells (SRBC) were obtained from Sclavo (Siena, Italy), and 4-hydroxy-3-nitrophenyl acetyl-O-succinimide (NP-O-Su) was purchased from Cambridge Research Biochemicals (Hariston, UK). NP-conjugated HRBC and SRBC were prepared as follows (7). Two milliliters of washed and packed RBC was suspended in 8 ml of borate-buffered saline, pH 9. NP-O-Su (20 mg in 2 ml of dimethylformamide) was added dropwise. After incubation for 10 min the reaction was stopped by addition of 40 ml of cold phosphate-buffered saline (PBS), pH 7.7, and RBC were washed three times in PBS before use. NP-conjugated syngeneic spleen cells (NP-SC) were prepared as described (7). Briefly, spleen cells from young mice (in one experiment, from old mice) sacrificed immediately after total body exposure to 2000 rad of X-rays were suspended in balanced salt solution (BSS) and adjusted to the desired cell concentration. After centrifugation, the pellet expected to contain 4 X lo8 nucleated cells was resuspended in 5 ml BSS 0. lx to lyse RBC. After 10 set 5 ml BSS 2x was added to prevent lysis of nucleated cells. After centrifugation the pellet was resuspended in 4 ml of PBS, pH 7.7, and then 100 ~1 of dimethyl sulfoxide, containing 2.4 mg of NP-O-Su, was added to the cell suspension. After 4 min 40 ml of BSS was added to stop the reaction, and, after centrifugation, NP-SC were washed twice in BSS and passed through a 100~pm nylon filter before use. Cyclophosphamide (CY) was purchased as Endoxan-Asta from Shering (Italy). Priming. Young and old mice were carrier-primed by iv injection of 2 X lo5 HRBC in 0.2 ml PBS to induce maximum numbers of helper T cells (8). After 4 days mice were sacrificed and their spleen cells were immunized in vitro with NP-HRBC. Responder cell cultures. Spleen cells from four HRBC-primed mice were suspended in complete culture medium containing RPM1 1640 (GIBCO Laboratories, Grand Island, NY) supplemented with 10% fetal calf serum (Flow Laboratories, UK), 2 mM rglutamine (GIBCO), and 10 pg/ml gentamicin (Shering, Kenilworth, NY), and cultured in 24-well tissue culture plates (No. 3524; Costar, Cambridge, MA) according to Mishell and Dutton (9). In each experiment triplicate culture wells received 0.5 ml medium containing 1 X 1O6NP-HRBC and 3 X 1O6nucleated spleen cells. Triplicate wells received no NP-HRBC and served as background controls. The anti-NP and
22
DORIA, MANCINI,
AND FRASCA
anti-HRBC antibody responses in each well were assayed on Day 4 of culture by the Cunningham and Szenberg technique (lo), with NP-SRBC or HRBC used as test antigens and evaluated as the number of direct plaque-forming cells (PFC) per culture well. For each antibody response the PFC background value, expressed by the geometric mean from triplicate wells without antigen, was subtracted from the number of PFC induced by antigen in each well. The geometric mean of net PFC numbers from triplicate wells was then calculated and reported as PFC per culture. An error factor by which the geometric mean should be multiplied or divided to obtain the variations due to one standard deviation is also given. Calculation of the geometric mean and its error factor was performed by antilog transformation of both the arithmetic mean of log PFC numbers from triplicate wells and its standard deviation. Induction of Tsl cells. NP-specific Tsl cells were activated in young and old mice by iv injection of 0.5 ml BSS containing 4 X lo7 NP-SC, prepared with SC from young mice. (In one experiment, young mice were given 4 X 105, 4 X 106, or 4 X lo7 NP-SC prepared with SC from young and old mice.) Six days later, four mice of each group were sacrificed and their spleen cells were used in vitro. TsFl-containing supernatant. This was a gift from D. H. Sherr, prepared from the B6-Ts l-29 hybridoma obtained by fusing BW 5 147 tumor cells with spleen cells from 2- to 3-month-old C57B1/6 mice (11). Subculture of Tsl and Ts2 cells. Generation of Ts2 cells in normal spleen cell populations is an antigen-independent process induced in culture by Tsl cells (12), activated in the spleens of NP-SC-injected mice, or by TsFl ( 13). Spleen cells from NPSC-injected mice and spleen cells from normal mice, hereafter designated Tsl cells and Ts2 cells, respectively, were mixed and subcultured in the same wells of Costar No. 3524 tissue plates under optimal conditions previously established for spleen cells from young and old mice (6). Mixing of Ts 1 cells with Ts2 cells was performed by combining spleen cells from mice of the same age (young or old). Unmixed Ts2 cells served as control. Each cell suspension was prepared from a pool of four spleens and adjusted to a concentration of 8 X lo6 nucleated cells/ml complete medium. Equal volumes of the Ts 1 and Ts2 cell suspensions were mixed so that 1 ml of the final suspension contained 4 X 1O6Ts 1 cells and 4 X 1O6Ts2 cells. Each final suspension of unmixed or mixed spleen cells was distributed in 4-24 wells (0.5 ml/well) and cells were cultured for 4 days. In some experiments, 0.5 ml of the unmixed Ts2 cell suspension from young or old mice was cultured with a monoclonal TsFl -containing supernatant which was added daily, in variable amounts (10 or 40 pl), to the Ts2 cell culture wells for 4 days. The addition of complete medium as control was used in preliminary experiments but was then abandoned because it had no effect. Thereafter, in one series of experiments cells harvested from the wells of each group were pooled, washed, centrifuged, resuspended in complete medium, and adjusted to a final concentration of 1.5 X lo7 nucleated cells/ml. Samples (0.1 ml) of each cell suspension were then added to responder cell culture wells 1 day before the PFC assays. In another series of experiments cell suspensions from the wells of each group were pooled and centrifuged, and their TsF2-containing supematant was collected and used immediately or frozen at -20°C. When used, graded aliquots of each supernatant were adjusted to a final volume, by adding complementary aliquots of complete medium, and then a constant aliquot, usually 300-500 ~1, of the final solutions containing different amounts of the original supematant was added to responder cell culture wells 1 day before the PFC assays.
AGING
AND SUPPRESSOR T CELLS
23
Ts3 cell depletion and enrichment. Depletion of Ts3 cell precursors was induced in young mice by CY treatment (13), as follows. One day after HRBC priming, mice were injected ip with 20 mg/kg CY in 0.2-0.25 ml PBS and, 3 days later, their spleen cells were used as responder cells in culture with NP-HRBC. Ts3 cell enrichment was performed by negative selection on plastic plates (Falcon, 3003 F) coated overnight with 5 ml of affinity-purified goat anti-mouse Ig antibody (100 &ml) and repeatedly washed with PBS before use (14). Briefly, responder spleen cells from HRBC-primed young or old mice were cultured with NP-HRBC for 3 days and then harvested, pooled, and transferred onto antibody-coated plates. Each plate received 5 X 10’ cells in 10 ml of complete medium without fetal calf serum and was incubated at 4°C for 1 hr. Nonadherent cells were recovered by gentle swirling and were usually more than 95% Thy-l .2+ and less than 5% Ig+, as detected by indirect fluorescence. This T cellenriched population was assumed to be enriched in Ts3 cells. Percentage of suppression of PFC responses. Suppression of anti-NP and antiHRBC PFC responses was evaluated by the geometric means of net PFC numbers from triplicate cultures that received either subcultured cells or their supernatants, as follows: Percentage suppression = 1 -
PFC (Tsl + Ts2) x loo PFC (Ts2) .
Ts 1 stands for Tsl cells or TsFl in subculture; Ts2 stands for Ts2 cells in subculture. An error factor, by which the percentage should be multiplied or divided to obtain the variations due to one standard error, was calculated by the antilog transformation of the standard error obtained by the application of Fieller’s theorem ( 15) to log values of geometric means and of their error factors. Estimates from independent experiments were combined by using log values of each percentage (Pi) and of its error factor (EFJ as follows. Each log Pi was multiplied by l/(log EFi)*, and the sum of log Pi/(lOg EFJ* was divided by the sum of l/(log EFJ* to provide the weighted mean of log P. The antilog transformation gives the weighted geometric mean of P. An error factor of the weighted mean was also calculated according to Cochran ( 16). Comparison among suppression percentages was performed by the t test using log values of the percentages and of their error factors. All data were computed as previously described ( 17). RESULTS Suppression by subcultured cells. Activation of Ts2 cells in subculture was induced by a monoclonal TsFl -containing supernatant that was obtained from a hybridoma originally derived from spleen cells of young mice. TsF 1 was used to replace Ts 1 cells and 10 or 40 ~1 of the supernatant was added daily to Ts2 cells in subculture for 4 days. Thereafter, subcultured cells were pooled and washed, and 1.5 X 1O6cells were added to 3-day cultures of responder cells from young mice. Each culture well was assayed on Day 4 for anti-NP and anti-HRBC PFC responses. Results in Table 1 demonstrate that if sufficient amounts of TsFl are added to subcultures, Ts2 cells from young mice but not Ts2 cells from old mice can be activated to transduce NPspecific suppression. Control subcultures indicate that the interaction between Tsl cells and Ts2 cells from old mice is very efficient, although to a lesser extent than the
24
DORIA, MANCINI,
AND FRASCA
TABLE 1 Age-Restricted Activation of Ts2 Cells That Transduce NP-Specific Suppression of Responder Cells from Young Mice Subculture EXPT. 1
2
3
Tsl cells
TsFl
Ts2 cells
Anti-NP PFC/culture
Young Old -
+ +
Young Young Young Old Old Old
1883(1.05) 2098 (1.02) 896(1.25) 1948(1.07) 1996(1.07) 1496 (1.07) 1956(1.12)
Young Old -
+ +
Young Young Young Old Old Old
3831 (1.27) 4244 ( 1.20) 1866(1.24) 2973 (1.35) 4598(1.18) 3084 (1.20) 4982(1.14)
Young Old -
+ +
Young Young Young Old Old Old
4102 (1.09) 5068 (1.20) 267 1 (1.05) 3315 (1.11) 3988 (1.12) 2827 (1.09) 3860(1.11)
w Suppression
51(1.45) 7 (1.05) 25 (1.06) 2(1.08)
56(1.17) 30(1.23) 33(1.15) 0
47(1.11) 35(1.13) 29 ( 1.09) 3(1.09)
Anti-HRBC PFC/culture 4501(1.09) 4911 (1.09) 4948(1.05) 4999 ( 1.05) 4840 (1.05) 4746(1.11) 4787 (1.05) 7527 (1.03) 8587 (1.05) 8081(1.05) 8109(1.05) 7817(1.03) 8181(1.12) 8360 (1.09) 1781 (1.05) 1772(1.07) 1747(1.05) 1790(1.09) 1751(1.09) 1832(1.09) 1727 (1.05)
% Suppression
0 0 2 (1.07) l(l.05)
6(1.05) 6(1.04) 0 0
l(l.06) 0 0 l(l.06)
Note. Spleen cells from young or old mice were subcultured in 0.5 ml of complete medium, as follows: 4 X 1O6Ts2 cells alone or with TsFl ; 2 X 106 Tsl cells and 2 X 1O6Ts2 cells. TsFl was added to subcultures daily, four times from Day 0 to Day 3: 10 pi/day in Experiment 1 or 40 pi/day in Experiments 2 and 3. On Day 4, subcultured cells were harvested, pooled, and washed, and 1.5 X lo6 cells were transferred to Day 3 responder cell cultures each of which was assayed for anti-NP and anti-HRBC PFC responses on the subsequent day. Anti-NP PFC/culture background responses without antigen: 5 14 (Experiment 1), I8 13 (Experiment 2), and 3 192 (Experiment 3). Anti-HRBC PFC/culture background responses without antigen: 220 (Experiment l), 176 (Experiment 2), and 110 (Experiment 3). Each background value (geometric mean from triplicate cultures) has been subtracted from the number of PFC induced by antigen in individual cultures. Geometric means of net PFC numbers from triplicate cultures are reported in the table. Numbers in parentheses are error factors by which means and percentages should be multiplied or divided to obtain variations due to one standard deviation and one standard error, respectively.
interaction between Ts 1 cells and Ts2 cells from young mice, confuming our previous findings (6). Suppression by subculture supernatant. A major advantage of using subculture supernatant rather than subcultured cells to transduce suppression is the possibility of adding increasing amounts of TsF2 to responder cells to attain maximum levels of suppression avoiding exceedingly high cell density in responder cell cultures. In this series of experiments 2 X lo6 Tsl cells and 2 X lo6 Ts2 cells from mice of the same age (young or old) were mixed in 0.5 ml/well and subcultured for 4 days. Unmixed Ts2 cells (4 X 106/0.5 ml) served as control. Thereafter, subculture supernatants were collected and added to responder cell cultures.
AGING
25
AND SUPPRESSOR T CELLS TABLE 2
Requirement of Cell Interaction for Generation of Subculture Supematant That Transduces Suppression of Responder Cells from Young Mice Cells in subculture Subcultured cells from mice Young
Ts 1 Tsl and Ts2
Subculture supematant (~1) added to responder cells
Anti-NP PFC per culture
% Suppression
-
2600(1.05) 2482 (1.03) 1055 (1.05) 1921 (1.05)
58 (1.04) 23 (1.03)
2349(1.02)
5 (1.03)
2336 (1.02) 1667 (1.05) 2149(1.02)
29(1.03) 8 (1.01)
2362 (1.02)
0
-
-
+
+
+ -
-
300 300 300
+
-+ -
-+ + -
150 150 I 300 300 300
+
--
-+
150 150 I
+ Old
Ts2
Note. Spleen cells from young or old mice were subcultured in 0.5 ml of complete medium, as follows: 4 X lo6 Tsl cells alone; 2 X lo6 Tsl cells and 2 X IO6 Ts2 cells; 4 X lo6 Ts2 cells alone. On Day 4, 300 ~1 of subculture supematant were transferred to Day 3 responder cell cultures which were assayed for antiNP PFC responses on the subsequent day. Anti-NP PFC/culture background response: 6 IO. The background value (geometric mean from triplicate cultures) has been subtracted from the number of PFC induced by antigen in individual cultures. Geometric means of net PFC numbers from triplicate cultures are reported in the table. Numbers in parentheses are error factors by which means and percentages should be multiplied or divided to obtain variations due to one standard deviation and one standard error, respectively.
Data from a preliminary experiment (Table 2) indicate that the supematant is very effective in transducing suppression if it is derived from a mixture of subcultured Tsl and Ts2 cells. Supematants from subcultures of unmixed cells have negligible effects, except for supematant from subcultured Tsl cells as expected from the fact that these spleen cells from NP-SC-injected mice comprise activated Tsl cells and Ts2 cells in unknown proportion. The data also show that the supematant activity depends on cell interaction in subculture because the pool of equal volumes of supematants from unmixed Ts 1 and Ts2 cells is devoid of sizable effects. It should be noted that supematants from subcultured cells from young mice are more effective than supematants derived from old mice when added to responder cells from young mice, suggesting that the interaction between TsF2 and Ts3 cells is age-restricted. The Tsl cell transfer from mice injected with NP-SC may convey NP-specific antibody-forming cells and antigen to subculture. However, the possibility that the supernatant activity is mediated by anti-NP antibodies produced in subculture is ruled out by the finding that comparable numbers of anti-NP PFC/spleen were found in young mice, either normal (360 PFC) or injected with 4 X 10’ NP-SC six days before sacrifice (420 PFC). Moreover, the same anti-NP antibody response was also found in 4day subcultures of 4 X lo6 mixed Tsl and Ts2 cells (890 PFC) or 4 X lo6 unmixed Ts2 cells (870 PFC) from young mice. In one experiment, 4 X 105, 4 X 106, or 4 X 10’ NP-SC, prepared with SC from young and old mice, were injected in young mice to induce Ts 1 cells. Mixed Ts 1 cells and Ts2 cells or unmixed Ts2 cells from young mice were subcultured for 4 days and
26
DORIA, MANCINI,
AND FXASCA
60
40
20
0 Subculture
supernatant
added
to
responder
cells
FIG. 1. Percent suppression of responder cells from young mice transduced by supernatants from subcultures containing Tsl and Ts2 cells from young mice. Tsl cells were activated by injection of 4 X lo5 (a q ), 4 X lo6 (A, A), or 4 X 10’ (0,O) NP-SC. Solid symbols refer to young SC donors, and open symbols refer to old SC donors. Error factors range from 1.02 to 1.06.
then their supernatants were transferred to responder cells from young mice 1 day before the anti-NP PFC assay. Figure 1 shows that suppression increases with the amount of the added supernatant up to a maximum level, reflecting the number of injected NP-SC and, therefore, the number of activated Ts 1 cells. No difference was found between the two NP-SC preparations, from young and old mice, suggesting that Ts 1 cell activation in young mice is not age-restricted. Age-related changes in the interaction between TsF2 and Ts3 cells were investigated as follows. In each experiment, Ts 1 cells and Ts2 cells from young or old mice were subcultured for 4 days and then graded amounts of the subculture supematants were added to responder cells from young or old mice. Using responder cells from young mice in two experiments and from old mice in four experiments it was found that after the addition of increasing amounts of supematant from subcultures containing Ts 1 cells and Ts2 cells from young or old mice the percentage suppression of the anti-NP PFC response increases up to plateau levels in all experiments (Fig. 2). However, using responder cells from young mice, suppression reaches a higher plateau level when the supematant is derived from young mice than when it is derived from old mice. Conversely, using responder cells from old mice, the two supematants bring about approximately the same plateau of suppression but the supematant derived from old mice is much more efficient than that derived from young mice. Hence, while 50 ~1 of the former supematant is sufficient to transduce maximum suppression, 300 ~1 of the latter is needed to reach the plateau level. The suppression profiles depicted in Fig. 2 demonstrate that the TsF2-mediated transduction of suppression of the anti-NP PFC response is age-restricted. The antigen specificity of suppression was assessed using responder cells from young or old mice and subculture supematant from young or old mice in optimal amounts to transduce maximum suppression. Results in Table 3 establish that sup pression is NP-specific and confirm the age restriction described in Fig. 2. Table 4 summarizes the results from four to seven experiments (pooled data from Fig. 2 and Table 3) showing not only that transduction of suppression is age-restricted but also that the same level of maximum suppression may be achieved under age-
AGING
27
AND SUPPRESSOR T CELLS
60 -
Responder
'
[
cells
F
100 Subculture
200 supernatant
300 added
fro+n
young
from
ild
mice
mice
400 to
responder
1
500
ul
cells
FIG. 2. Percent suppression of responder cells from young or old mice transduced by supematants from subcultures containing Tsl and Ts2 cells from young (0) or old (0) mice. Symbols represent weighted means from two to four experiments. Error factors range from 1.02 to 1.11.
matched conditions. Thus, the supematant-responder cell mixture yields equal levels of maximum suppression in the young-young and old-old combinations. The evidence so far presented for age-restricted transduction of suppression implies that TsF2 and Ts3 cells may be derived from mice of the same age but may also require age matching of responder B cells. This issue was investigated by use of TsF2containing supematant derived from old mice to transduce suppression of responder cells from young mice, after depletion of Ts3 cell precursors by in vivo CY treatment, and in vitro addition of activated Ts3 cells from old mice. Table 5 presents all data from one experiment. The efficiency of the CY treatment to deplete responder spleen cells of Ts3 cell precursors is shown by the difference in PFC response between groups I and VI as well as between the background control groups and by the negligible level of suppression in groups VIII and X. The efficiency of adding activated Ts3 cells from young or old mice to reconstitute responder cell populations sensitive to subculture supematants is demonstrated by the same loss (50%) of suppression from groups III (58%) to V (30%) as that from groups XII (47%) to XIV (24%) and by the comparable levels of suppression observed in groups XII (48%) and XVI (46%) as expected from data in Fig. 2. The critical result ofthis experiment, however, is the difference in percentage suppression between groups XIV (24%) and XVIII (52%). Althougb B cells in group XVIII are from young mice, the replacement of Ts3 cells from young mice with Ts3 cells from old mice increases the sensitivity of the responder cells to the subculture supematant derived from old mice up to the same level exhibited by responder cells from old mice (Fig. 2 and Table 4). It appears, therefore, that age matching between TsF2 and Ts3 cells is necessary and sufficient for effective suppression of responder cells. The experiment was repeated and provided similar results. DISCUSSION This work extends our previous investigation on the effects of aging on immunosuppression (6) and indicates the NP system as a reliable and suitable experimental
28
DORIA, MANCINI,
AND FRASCA
TABLE 3 NP Specificity and Age Restriction of Maximum Suppression Transduced by Subculture Supematant
EXPT 1
2
3
4
5a
5b
Responder cells from mice Young
Young
Young
Young
Young
Old
Subculture Ts 1 cells
-
Ts2 cells
-
Anti-NP PFcfculture
Young Old
Young Young Old Old
4176 (1.07) 4173 (1.09) 2245 (1.16) 4189(1.12) 2697 (1.16)
Young Old
Young Young Old Old
4860(1.20) 4762 (1.22) 1960(1.14) 4799(1.14) 2579 (1.09)
Young Old
Young Young Old Old
7418(1.05) 7262(1.05) 3556 (1.05) 7615 (1.02) 4465 (1.03)
Young Old
Young Young Old Old
3296 (1.09) 3166(1.09) 1508 (1.16) 3278 (1.03) 1967(1.11)
Young Old
Young Young Old Old
2312(1.07) 2324 (1.07) 1079(1.18) 2328 (1.03) 1231(1.22)
Young Old
Young Young Old Old
505 (1.12) 498 (1.27) 304 ( 1.20) 520(1.07) 263(1.24)
96 Suppression
Anti-HRBC PFC/culture
% Suppression
46(1.11)
2366(1.02) 2337(1.07) 2379(1.03) 2312(1.03) 2336 ( 1.07)
0
4070 (1.03) 3895 (1.05) 4004(1.03) 3947 (1.05) 3951(1.02)
0
36(1.11)
59(1.15) 46(1.10)
51(1.05) 41(1.02)
52(1.10) 40(1.06)
54(1.11) 47 (1.41)
39 (1.20) 49(1.14)
0
0
1228 (1.05) 1246(1.09) 1229(1.02) 1255 (1.03) 1260(1.07)
l(l.05)
1388(1.05) 1368 (1.05) 1309 (1.03) 1299 (1.05) 1282 (1.64)
4(1.03)
1681 (1.02) 1687 (1.03) 1708(1.12) 1868 (1.14) 1727 (1.03)
0
525 (1.09) SOS(l.16) 585 (1.09) 565(1.11) 612 (1.09)
0
l(l.04)
8 (1.09)
0 0
Note. Spleen cells from young or old mice were subcultured in 0.5 ml of complete medium, as follows: 2 X lo6 Tsl cells and 2 X lo6 Ts2 cells, or 4 X lo6 Ts2 cells alone. On Day 4, subculture supematants were collected and transferred to Day 3 responder cell cultures. Cultures in all groups received 300 ~1 of supematant except in the last two groups of Experiment 5b in which responder cell cultures received 50 ~1 of supematant. Each responder cell culture was assayed for anti-NP and anti-HRBC PFC responses on Day 4. Anti-NP PFC/culture background responses without antigen: 776 (Experiment l), 639 (Experiment 2), 1318 (Experiment 3), 799 (Experiment 4), 457 (Experiment 5a), and 225 (Experiment 5b). Anti-HRBC PFC/culture background responseswithout antigen: 366 (Experiment 1), 101 (Experiment 2), 197 (Experiment 3), 130 (Experiment 4), 78 (Experiment 5a), and 66 (Experiment 5b). Each background value (geometric mean from triplicate cultures) has been subtracted from the number of PFC induced by antigen in individual cultures. Geometric means of net PFC numbers from triplicate cultures are reported in the table. Numbers in parentheses are error factors by which means and percentages should be multiplied or divided to obtain variations due to one standard deviation and one standard error, respectively.
AGING
29
AND SUPPRESSOR T CELLS TABLE 4
Maximum Suppression of Responder Cells Transduced by Subculture Supematant 96 Suppression of responder cells from mice Subcultured cells from mice
yaw!
Old
Young
Old
P
Note. Percentages are weighted means from 4 to 7 experiments. Error factors range from 1.02 to 1.06. NS stands for not significant at P = 0.60.
model for quantitative studies of the molecular and cellular elements involved in the induction, transduction, and effector phase of antigen-specific suppression. The number of activated Tsl cells depends on the NP-SC dose as larger doses of injected NP-SC yield subculture supernatants with higher efficiency in transducing suppression when added in graded amounts to responder cell cultures (Fig. 1). Since a plateau level of suppression was attained for each NP-SC dose, this finding suggests the existence of Ts 1 cell subsets with different thresholds of activation, each of which is recognized by a specific Ts2 subset that, in turn, triggers a complementary Ts3 cell subset. Different Id and anti-Id receptors may be the recognition elements of the different cell subsets. The data also show that NP-SC derived from young or old mice induce the same levels of suppression of responder cells from young mice. Thus, whatever the mechanism of antigen presentation to Ts cells in the induction phase ( 1, 1g), activation of Ts 1 cells is not age-restricted. Activation of Ts2 cells by Ts 1 cells is mediated by Id+ TsFl recognized by anti-Id Ts2 cell receptors ( 1, 2). Activation of Ts2 cells from young, but not from old, mice is induced in subculture by a monoclonal TsFl-containing supematant obtained from a hybridoma originally derived from spleen cells of young mice (Table 1). This age restriction is in keeping with our previous finding of a poor interaction in subculture between Tsl cells from young mice and Ts2 cells from old mice (6) and suggests that age mismatching negatively affects the idiotypic recognition involved in Ts2 cell activation. The data are compatible with two possibilities. (i) The idiotypic repertoire undergoes qualitative changes with aging and therefore the Id+ TsFl produced by Tsl cells from young mice is not recognized by anti-Id Ts2 cells from old mice. (ii) If there are subsets of Tsl cells and Ts2 cells characterized by different Id and anti-Id receptors, the loss of a Ts2 cell subset in old mice may completely impair the recognition ofthe monoclonal TsFl produced by a Tsl cell subset from young mice. Possibility (ii) is favored by the results in Table 1 and Fig. 1. The interaction between TsF2-containing subculture supematant and the Ts3 cells activated by antigen in responder cell culture is age-restricted (Fig. 2). Suppression of responder cells from young mice attains a higher plateau level when the subculture supematant is derived from young rather than from old mice. Conversely, suppres-
30
DORIA, MANCINI,
AND FRASCA
TABLE 5 Age-Related Changes in the Interaction between Subculture Supematant and Ts3 Cells Subculture Group
Tsl cells
I II III IV V VI VII VIII IX X XI XII XIII XIV xv XVI XVII XVIII
Young Old Young Old Young Old Young Old
Responder cells from young mice
Ts2 cells
CY treatment
Young Young Old Old Young Young Old Old Young Young Old Old Young Young Old Old
+ + + + + + + + + + + + +
Added Ts3 cells
Young Young Young Young Old Old Old Old
Anti-NP PFC/culture 1015 (1.03) 981(1.05) 408 (1.03) 955 (1.02) 668 (1.05) 1267 (1.03) 1214(1.02) 1129(1.05) 1267(1.02) 1186(1.05) 1267(1.02) 665 (1.09) 1240 ( 1.03) 937(1.11) 1265 (1.05) 681 (1.18) 1220(1.02) 591(1.12)
% Suppression
58 (1.04) 30(1.03) 7 (1.03) 6(1.03) 47 (1.05) 24(1.07) 46(1.11) 52 (1.07)
Note. Spleen cells from young or old mice were subcultured in 0.5 ml of complete medium, as follows: 2 X lo6 Tsl cells and 2 X lo6 Ts2 cells, or 4 X IO6 Ts2 cells alone. On Day 4, subculture supematants were collected and 300 pl was transferred to responder cell cultures, except groups I and VI, 1 day before the anti-NP PFC assay.Responder spleen cells (3 X 106) from HRBC-primed and CY-treated (+) or untreated (-) young mice were cultured with NP-HRBC for 4 days and then assayed for the anti-NP PFC response. On Day 3, cultures of Ts3 cell precursor-depleted responder cells from CY-treated mice received no further cells (groups VI-X) or 2 X 10’ cells, after enrichment in T cells including NP-activated Ts3 cells, from 3 day cultures containing NP-HRBC and responder cells from HRBC-primed young (groups XI-XIV) or old (groups XV-XVIII) mice. Anti-NP PFC/culture background responses without antigen: 271 for cells from CY-untreated mice and 339 for cells from CY-treated mice. Each background value (geometric mean from triplicate cultures) has been subtracted from the number of PFC induced by antigen in individual cultures of cells from CY-untreated or CY-treated mice. Geometric means of net PFC numbers from triplicate cultures are reported in the table. Numbers in parentheses are error factors by which means and percentages should be multiplied or divided to obtain variations due to one standard deviation and one standard error, respectively.
sion of responder cells from old mice reaches approximately the same plateau level when the subculture supernatant is derived from young or old mice, but the supematant from old mice is much more efficient than that from young mice when added in small amounts. If the maximum levels of suppression are compared (Table 4) transduction of suppression appears to be equally effective when age-matched TsF2 and Ts3 cells are used, both from young and old mice. These data are compatible with the existence of different anti-Id TsF2 subsets which specifically recognize complementary Id+ Ts3 cell subsets. The loss of one or more TsF2 and Ts3 cell subsets in old mice may explain why the subculture supematant derived from old mice is less efficient than the supematant from young mice to transduce maximum suppression of responder cells from young mice and why the supematant from old mice is at least as effective as the supernatant from young mice to maximally stimulate Ts3
AGING
31
AND SUPPRESSOR T CELLS
Ts3
Ts2
Tsl (LYt-1)
(Lyt-12)
TsFl
w
(Lyt-2)
TsF2
.
FIG. 3. A minimal model for age-related changes in Ts cell subsets of inducer (Tsl), transducer (Ts~), and effector (Ts3) cell populations of the NP-specific suppressor circuit. 0 and n represent idiotypes associated with anti-NP receptors.
cells from old mice. The high efficiency of the small amounts of subculture supernatant from old mice to transduce suppression of responder cells from old mice suggests that affinity of the anti-Id TsF2 and Id+ Ts3 cell reaction increases with aging. This feature may also explain why small amounts of subculture supernatants from young or old mice are equally effective in transducing suppression of responder cells from young mice although old mice have lost Ts2 and Ts3 cell subsets. The possibility of an age-related increase in Ts cell Id anti-Id binding affinity need not be surprising since high-affinity B cell clones do not disappear with aging, but are simply masked by auto-anti-Id (high affinity) antibodies ( 19). Age matching between TsF2 and Ts3 cells is necessary and sufficient for effective suppression of responder cells (Table 5), suggesting that mismatching between Ts3 and B cells is irrelevant. Thus, age restriction is operating within a suppressor T cell circuit, without affecting antigen presentation to Tsl cells or the interaction between Ts3 cells and target B cells. The model proposed in Fig. 3 offers an interpretation for the changes in antigenspecific immunosuppression observed in aging mice. The data are more easily explained by subset deletions in Ts 1, Ts2, and Ts3 cell populations rather than by qualitative modifications of the Ts idiotypic repertoire. The latter possibility might result from intrinsic accumulation of mutations or epigenetic errors (20) or be secondary to changes in the B cell Id anti-Id repertoire (2 l), some of which are known to occur in aging (3). The loss of Ts cell subsets may be a consequence of thymus involution which has been shown to decrease several T cell functions (22). However, studies on Ts cells have yielded controversial results as immunosuppression appears to increase (23-24), decrease (3 1, 32, 34-39), or remain unchanged (29) in aging mice and humans. This controversy reflects the large variety of experimental models and methods used which may deal with nonidentical parts of the same or different Ts cell circuits and, therefore, are likely to provide dissimilar results. The present analysis of the NPspecific Ts cell circuit suggests that aging affects immunosuppression by loss of Ts cell subsets. Yet, under age-matched conditions, transduction of suppression is more effective when subculture supematant and Ts3 cells are derived from old rather than
32
DORIA, MANCINI,
AND FRASCA
from young mice while the same level of maximum suppression is attained. This observation is consistent with the possibility that higher affinity of the interactions involved in Ts cell activation compensates to some extent for the loss of Ts cell subsets in old mice. Furthermore, it is conceivable that during senescence a greater inducibility of Tsl cells may help to counteract the effects of a decrease in Ts cell subsets that results from thymus involution. This possibility, previously shown to occur for the azobenzenearsonate system (33), is presently under investigation for the NP system in our laboratory. REFERENCES 1. Dorf, M. E., and Benacerraf, B., Annu. Rev. Immunol. 2,127, 1984. 2. Asherson, G. L., Colizzi, V., and Zembala, M., Annu. Rev. Immunol. 4,37, 1986. 3. Goidl, E. A., Thorbecke, G. J., Weksler, M. E., and !&kind, G. W., Proc. Natl. Acad. Sci. USA 77, 6788,198O.
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