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Age-dependent changes of B-cell reactivity and T cell-T cell interaction in the in vitro antibody response
CELLULAR
IMMUNOLOGY
53, 195-206 (1980)
Age-Dependent Changes of B-Cell Reactivity and T Cell-T Interaction in the in Vitro Antibody Response’ GINO DORIA, CNEN-Euratom
GIACOMO
D’AGOSTARO,
Cell
AND MAURO GARAVINI
Immunogenetics Group, Laboratory of Radiopathology, CSN Casaccia, Rome, Italy Received October 25, 1979
The in vitro anti-2,4,6-trinitrophenyl (TNP) antibody response of spleen cells from 1- to 32-month-old mice was evaluated by techniques suitable for dissecting out T- and B-cell reactivities. Spleen cells from individual mice carrier primed with horse red blood cells (HRBC) were immunized in vitro with TNP-HRBC, a T-dependent immunogen. The anti-TNP antibody response was found to change with the increasing age at which the animals were carrier primed, so that the response at 24 months was only about 4% of that at 3 months of age. It was also found that the variability in response among mice of the same age increased exponentially with age, suggesting that during senescence the immune system is impaired by stochastic events. A substantial part of the decline in immune responsiveness reflects the age-dependent decrease of B-cell reactivity as shown by marked reduction of the anti-TNP antibody response of spleen cells from old mice uponin vitro immunization with TNP-Ficoll, a T-independent immunogen. To analyze helper T-cell activity of aging mice independently from age-associated changes in B-cell reactivity the helper cell function was titrated by adding graded numbers of HRBC-primed spleen cells from individual mice of a given age to cultures containing a constant number of normal spleen cells from a pool of 3-month-old mice and the immunogen TNP-HRBC. The anti-TNP antibody response increases with the number of primed cells added to culture and the log number of anti-TNP plaque-forming cells is a linear function of the log number of the primed cells added. This linear regression was used to evaluate T cell-T ceil interactions. This analysis unmasked in aging mice a decrease in the ability of T cells to interact synergistically in the generation of helper activity.
INTRODUCTION In several animal species the antibody response increases during postnatal development, reaches a maximum in adulthood, and then declines during senescence. Changes in the population of immunologically competent cells and their functional consequences account for most of the variation of immunological competence (1). Thus, the decline of immune responsiveness in old mice is compatible with a decrease in the size and reactivity of the helper T-cell population (2, 3), an increase in the suppressor T-cell population (3-6), or both. Thymic involution, which is a prominent phenomenon during senescence, points to changes in T-cell populations as an important cause of the decline in immune functions. r This work was supported by a CNEN-Euratom Biology Division.
contract. Publication No. 1610 of the Euratom
195 0008-8749/80/090195-12$02.00/O Copyright Q 1980by AcademicPress,Inc. AU rights of reproduction in any form reserved.
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DORIA,
D’AGOSTARO,
AND GARAVINI
Alterations of the B-cell population have also been described in aged animals (1,3, 7). Macrophages seem to be unaffected by aging (8-10). We have previously described a parabolic rise and fall of the antibody response and affinity in aging mice, which have been attributed mainly to changes in type and degree of the cell interactions involved in the immune response (11). In the present study, a cellular analysis of age-dependent variations of the in vitro antibody response was carried out by techniques suitable to dissect out T- and B-cell reactivities. Functions of both T and B cells were found to change with aging. MATERIALS
AND METHODS
Animals and their carrierpriming. Male (C57BVlO x DBA/2) F, mice were used when 1-32 months old. For induction of helper cells, mice of a given age were carrier-primed by injecting iv 2 x lo5 (if not otherwise specified) horse red blood cells (HRBC)’ in 0.2 ml PBS 4 days before sacrifice. Red blood cells. HRBC and SRBC were prepared from blood suspended in Alsever’s solution, purchased from Sclavo (Siena, Italy), and washed repeatedly with PBS. TNP-RBC. TNP-HRBC were prepared by heavy coupling of TNBS to HRBC (12) and used as a T-dependent immunogen. TNP-SRBC were prepared by light coupling of TNBS to SRBC (13) and used as test antigen in the Jerne technique to detect direct anti-TNP PFC. These hemolytic plaques were shown to be TNP specific by inhibition with soluble TNP-bovine y-globulin or TNP-•-aminocaproic acid (14). TNP-Ficoll. TNBS was conjugated to Ficoll (MW 400,000) from Pharmacia (Uppsala, Sweden) according to Inman (15). The conjugate TNP-Ficoll with a molar ratio of 79: 1 was used as T-independent immunogen. Cell culture. Spleen cell suspensions were prepared from normal or carrierprimed mice and cultured by the method of Mishell and Dutton (16) using microtissue culture plates (Falcon No. 3040, Oxnard, Calif.) as previously described (17). Culture medium was Eagle’s minimum essential medium (Microbiological Associates, Bethesda, Md.) supplemented with 10% fetal calf serum (Rehatuin, Reheis, Phoenix, Ariz.), 1 miV sodium pyruvate, 0.1 m&f nonessential amino acid mixture, 100 IU/ml penicillin, 100 pg/ml streptomycin sulfate, 2 mkf L-glutamine (all from Microbiological Associates, Bethesda, Md.), and 20 m&f Hepes buffer solution (Eurobio, France). No mercaptoethanol was added to culture. This culture system was used to evaluate in vitro antibody responses to T-dependent and T-independent immunogens and to titrate in vitro helper cell activity, as described below. In vitro antibody response. The anti-TNP antibody response to a T-dependent or T-independent immunogen was elicited by immunizing spleen cells from carrierprimed or normal mice with TNP-HRBC or TNP-Ficoll, respectively. The immunization conditions were the following: 1 x lo6 nucleated spleen cells in 0.1 ml medium containing 2 x lo5 TNP-HRBC or 1.5 x 10” nucleated spleen cells in 0.2 ml medium containing 20, 2, or 0.2 ng TNP-Ficoll. After 4, 5, and 6 days of 2 Abbreviations used: HRBC, horse red blood cells; SRBC, sheep red blood cells; PBS, phosphatebuffered saline; TNP, 2,4,6&initrophenyl; TNBS, 2,4,6-trinitrobenzensulfonic acid; PFC, plaqueforming cells.
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culture the cells were harvested from four to eight wells of the same experimental group, pooled, washed, and assayed for direct anti-TNP PFC. The response was evaluated by the number of anti-TNP PFC per culture well. In the experiments with TNP-HRBC the number of PFC per spleen was calculated as PFC per culture x total nucleated cells per spleen. The results are presented as the peak responses found on Days 4 and 5 for TNP-Ficoll and TNP-HRBC, respectively. The T dependence or T independence of the response to the immunogens used was verified in preliminary experiments as follows. The anti-TNP antibody response of HRBC-primed spleen cells immunized with TNP-HRBC was shown to be impaired by pretreatment of the primed cells with mouse anti-Thy 1.2 antiserum and rabbit C. This cytotoxic treatment, however, did not affect the anti-TNP antibody response of normal spleen cells immunized with TNP-Ficoll. In each experiment with carrier-primed spleen cells and TNP-HRBC, the spleen cells were derived from 5 or 6 individual mice of a given age and from a pool of 3-month-old mice (5 animals). Since mice of different ages were tested in different experiments, the response of the spleen cells from each mouse was expressed as the percentage of the response of the pooled spleen cells used in the sameexperiment as reference age control. When normal spleen cells were immunized with TNP-Ficoll, pooled mice (8- 10 animals/group) of different ages were compared in the same experiment and the anti-TNP responses could be expressed directly as numbers of PFC per culture well. Titration of in vitro helper cell activity. A modification of the experimental model of Kettman and Dutton (18) was used to assay the level of helper cell activity in mice carrier primed at different ages. Normal spleen cells immunized in vitro with TNP-HRBC respond poorly to TNP. If graded numbers of spleen cells from HRBC primed mice are added to a constant number of normal spleen cells the anti-TNP response increases with the number of primed cells in the range where helper activity is limiting (17). If carrier-primed spleen cells from mice of different ages are added to normal spleen cells from mice of a constant age the evaluation of helper activity is independent from age-associated changes in B-cell reactivity which would affect in vivo estimates. In each experiment, all culture wells received 0.1 ml medium containing 2 x lo5 TNP-HRBC and 1 x IO6nucleated normal spleen cells from a pool of 3-month-old mice (lo- 15 animals) and were divided into three groups according to the addition of HRBC-primed cells. These cells were sampled from the same spleen suspensions also used in parallel cultures to determine the anti-TNP response as described in the preceding section. Culture wells of one group received no additional cells and served as baseline controls. Culture wells of the other two groups in addition received graded numbers (5-40 X 104) of nucleated spleen cells either from individual HRBC-primed mice of a given age or from a pool of HRBC-primed 3-month-old mice (5 animals) which served as reference age control. Cells from the same four to eight wells were harvested on Day 4,5, and 6 of culture and assayed for the number of direct anti-TNP PFC per culture well. The peak responses were found on Day 5 and processed as follows. The anti-TNP response of the normal spleen cells, which was very low (less than 10% of the response of 1 x lo6 nucleated spleen cells from a pool of carrier-primed 3-month-old mice), was subtracted from the responses of the normal spleen cells supplemented with primed cells. The net response was found to increase with the number of primed cells added to culture, so that the log number of
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DORIA, D’AGOSTARO, AND GARAVINI
PFC was a linear function of the log number of primed cells added. Linear regressions were calculated for spleen cells from individual mice of a given age and for the pooled spleen cells from 3-month-old mice. The regression coefficient, which should represent the number of interacting cell populations (11, 19), was taken as an estimate of the degree of T cell-T cell interactions within the population of carrier-primed spleen cells that help the response of normal spleen cells. In control experiments, pooled spleen cell suspensions from carrier-primed 3-month-old mice were filtered through glass wool columns (20) before culture. Statistical analysis. Linear regressions and correlations were calculated as described by Steel and Torrie (21). Polynomial regressions were fitted by a previously described method (22). RESULTS Dose of Carrier Priming The optimal dose of HRBC to carrier-prime mice of different ages was determined in a preliminary experiment in which five mice at the ages of 1,3, or 20 months were injected with 2 x 104-2 x IO6HRBC. The animals were sacrificed 4 days later and pooled spleen cells from the carrier-primed mice of each group and from five unprimed age-control mice were immunized in vitro with 2 x IO5 TNP-HRBC. The results in Table 1 show that the anti-TNP peak response of spleen cells, expressed as PFC per culture or PFC per spleen, is greatly enhanced by carrier priming and that 2 x lo5 HRBC is the optimal priming dose at the three ages investigated. This dose was chosen for the subsequent experiments. TABLE 1 Effect of HRBC Priming on the in Vitro Anti-TNP Antibody Response of Spleen Cells from Aging Mice Age (months)
HRBC
PFCkulture
PFC/spleen
1
0 2 x 104 2 x 105 2 x log
16 110 248 193
1,352 9,626 32,552 21,203
3
0 2 x 104 2 x 105 2 x 106
26 553 888 508
4,762 99,440 157,318 107,925
20
0 2 x 104 2 x 105 2 x 106
14 13 511 108
2,116 1,706 66,021 13,594
Note. Spleen cells from mice unprimed or primed with different numbers of HRBC were cultured with 2 x 1W TNP-HRBC. Peak responses obtained at Day 5 of culture are referred to 1 X 106 nucleated cells initially put in culture. The number of anti-TNP PFCkulture was multiplied by the total number of nucleated cells/spleen to obtain the number of PFC/spleen.
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In Vitro Antibody Response Mice at the age of l-27 months were primed with 2 x lo5 HRBC and, 4 days later, individually tested for the ability of their spleen cells to mount an in vitro anti-TNP antibody response upon immunization with TNP-HRBC. The results of 14 experiments, in each of which five or six mice were individually tested, are reported in Fig. 1. The anti-TNP response of the spleen cells from each mouse is expressed as percentage of the response of the pooled spleen cells from 3-month-old mice used in the same experiment as reference age control. The upper part of Fig. 1 illustrates that after the initial rise from 1 to 3 months, the antibody response declines with increasing age so that at 24 months it is about 4% of that at 3 months.
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FIG. 1. In vitro anti-TNP antibody response of HRBC-primed spleen cells Born aging mice upon immunization with TNP-HRBC. (Top) Each point represents the response of spleen cells from a single mouse of a given age expressed as a percentage of the response of spleen cells from a pool of 3-month-old mice (reference age control). The number of PFC refers to 1 x 109nucleated cells initially put in culture. The line represents a second-order polynomial regression fitting all points including a zero value (omitted from the semilog diagram) of a 27-month-old mouse. (Bottom) Coefficient of variability: 100 x SD/ mean. The line represents the best-fitting regression calculated by the least-squares method.
200
DORIA,
D’AGOSTARO,
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Furthermore, this decline with age is concomitant with an increasing spread of the responses of individual mice. The lower part of Fig. 1, indeed, shows that the variability of the response increases exponentially with age, as evidenced by a statistically significant (P < 0.01) correlation (r = 0.82) between the log coefficient of variability and age. The decrease in antibody response of 1 x lo6 nucleated cells in culture could result from age-dependent alterations of the spleen cell populations such as diminution of the absolute number of immunologically competent cells or presence of abnormal (malignant) cells diluting out the number of competent cells in the sample tested in culture. The latter possibility might have occurred in 5 mice in which the range in total numbers of nucleated spleen cells was 2.9-9 x 108. However, the spleens of the other 66 mice contained 1-25 x lo8 nucleated cells, a range in cell numbers too narrow to account for the observed variation in antibody response. The possibility that the decline in response reflects an absolute decrease in the spleen pool size of immunologically competent cells is suggested by the demonstration in Fig. 2 that: (a) the response of 1 x lo6 nucleated spleen cells from all 71 mice is not correlated (r = 0.18;P > 0.05) with the total number of nucleated spleen cells (Fig. 2, right); (b) the response of 1 x lo6 nucleated spleen cells not only is correlated (r = 0.75; P < 0.01) with the response per spleen but, if the most extreme point (highest number of PFUspleen) is disregarded, the linear regression coefficient f SE is 0.93 + 0.04, avalue very close to 1 as expected if the spleen size
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FIG. 2. Effect of spleen size on the in vitro anti-TNP antibody response of HRBC-primed spleen cells from aging mice upon immunization with TNP-HRBC. Each point represents the response of spleen cells from a single mouse of any age expressed as a percentage of the response of spleen cells from a pool of 3-month-old mice (reference age control). The number of PFC refers to 1 x 106nucleated cells initially put in culture. (Lefr) Positive correlation between PFC/culture and PFClspleen. (Right) Lack of correlation between PFC/culture and nucleated cells/spleen.
201
AGING OF B- AND T-CELL IMMUNE FUNCTIONS
does not affect the in vitro response (Fig. 2, left). However, it cannot be excluded that the decline in response reflects qualitative rather than quantitative abnormalities of immunologically competent cells or a combination of the two possibilities. The age-dependent decline of the in vitro antibody response of carrier-primed spleen cells to a T-dependent antigen such as TNP-HRBC is compatible with alterations of the T- and B-cell populations. Defects in the B-cell population were demonstrated by the finding of a decrease in the anti-TNP response of normal spleen cells from old mice immunized in vitro with TNP-Ficoll, a T-independent antigen. The results of the experiment reported in Fig. 3 describe an increase of the response from 1 to 5 months of age followed by a continuous decrease until 32 months. This parabolic variation of the response with aging was consistent at three antigen doses. Titration of In Vitro Helper Cell Activity The helper function of T cells was titrated by adding graded numbers of carrier-primed spleen cells from individual mice of a given age to cultures of a constant number of normal spleen cells from a pool of 3-month-old mice. Upon immunization with TNP-HRBC, the in vitro anti-TNP antibody response of the normal spleen cells increased with the number of added carrier-primed spleen cells. Figure 4 describes the results of an experiment in which five individual mice were tested at the age of 3 months. When the linear regressions (log PFCkulture on log primed cells added to culture for individual mice) are compared to the regression for the reference age control (carrier-primed spleen cells from a pool of five mice at the age of 3 months) the straight lines for individual mice overlap to a great extent with the control line as expected, although some individual variation is also evident. The
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FIG. 3. In vitro anti-TNP antibody response of normal spleen cells from aging mice upon immunization with TNP-Ficoll. Each point represents a pool of 8- 10 mice of a given age.
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FIG. 4. Titration of in vitro helper cell activity by the linear regression log PFCkulture on log primed spleen cells added to cultures of normal spleen cells from a pool of 3-month-old mice. Straight lines for primed spleen cells from five mice individually tested at 3 months are compared to the line (dashed) for the primed spleen cells from a pool of five mice tested at 3 months (reference age control). The lines represent best-fitting regressions calculated by the least-squares method. Slopes are represented by b values.
results of another experiment reported in Fig. 5 illustrate that the slopes of the straight lines for five individual mice tested at the age of 24 months are always lower than those for the reference age control, indicating a striking defect in helper cell activity. Linear regressions (log PFC/culture on log primed cells) were calculated for 71 mice individually tested at different ages in 14 experiments as well as for the pool of 3-month-old mice used in each experiment as reference age control. The mean slope 4 SE of the linear regressions for the reference age controls from all experiments is 1.38 ? 0.08. Since the standard error is very small (6%) the regression coefficients for individual mice tested in different experiments are reported in Fig. 6 without normalization to the reference age controls. The data indicate age-dependent alterations in type and degree of T cell-T cell interaction within the carrier-primed spleen cell population. Positive cooperation between two T-cell subpopulations is evident at l- 10 months (coefficient > l), negligible at 1I- 12 months (coefficient = I), and inhibited at 15, 18, 21, 24, and 27 months (coefficient < 1). The interpretation of the titration data on helper cell activity is based on the assumption that B cells and macrophages of the carrier-primed cell population do not contribute to the anti-TNP response of normal spleen cells. The irrelevance of
203
AGING OF B- AND T-CELL IMMUNE FUNCTIONS AGE:
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FIG. 5. Titration of in virro helper cell activity by the linear regression log PFCkulture on log primed spleen cells added to cultures of normal spleen cells from a pool of 3-month-old mice. Straight lines for primed spleen cells from five mice individually tested at 24 months are compared to the line (dashed) for the primed spleen cells from a pool of five mice tested at 3 months (reference age control). The lines represent best-fitting regressions calculated by the least-squares method. Slopes are represented by b values.
the B-cell component of the primed cells was demonstrated in previous experiments (17) by the same anti-TNP response of 1 X lo6 normal spleen cells cultured with 30 x lo4 HRBC-primed spleen cells untreated or pretreated with rabbit anti-mouse Ig antiserum and guinea pig C, a treatment shown to be very effective in abrogating the in vitro primary response of normal spleen cells to SRBC. The contribution of macrophages was ruled out by the demonstration that efficient removal of macrophages from the carrier-primed spleen cell population by two passages through glass wool does not affect helper cell activity (Table 2). DISCUSSION The analysis of T- and B-cell reactivities as carried out in the present study revealed new and marked alterations in the lymphocyte populations of aging mice, implementing and clarifying previous results from in vivo and in vitro studies on the senescence of the immune system (l-3,9,11,23-27). The changes in T- and B-cell reactivities as evidenced by the present analysis might have resulted from age-dependent alterations in cell replication capacity arising in vitro rather than preexisting in vivo . However, since the same changes in percentage replicating cells and cell population doubling time have been shown to occur both in vitro and in vivo
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DORIA, D’AGOSTARO, AND GARAVINI
T 0 1
T 0 1
2
FIG. 6. Changes in T cell-T cell interactions with aging. Each point represents the mean slope of linear regressions log PFClculture on log primed spleen cells from 5 to 10mice individually titrated for in virro helper cell activity. Vertical bars denote -C1SE. The dashed line is positioned at 1 which is the expected slope value when no interaction occurs within the carrier-primed spleen cell population.
(28), the decline of in vitro immune functions with increasing age can be attributed to in vivo cell alterations. The anti-TNP antibody responsiveness of spleen cells immunized with a T-independent antigen was found markedly decreased in old mice. Defects in the B-cell population appear after 5 months of age as evidenced by a IO-fold reduction in the antibody response to TNP-Ficoll (Fig. 3). The extent of impairment of this response is at variance with the only slight decrease observed in the mitotic responsiveness of B lymphocytes to lipopolysaccharide (1, 26, 29) and with the finding that the population size of B lymphocytes, as detected by surface markers, is not decreased in old mice (30). Thus, qualitative abnormalities in the B-cell population seem to play a greater role than cell depletion (3). TABLE 2 Persistent Helper Cell Activity of Carrier-Primed Spleen Cells after Macrophage Depletion Log PFC vs log primed cells regression Passage on glass wool None 1 2
PFCkulture 875 825 163
Intercept
Slope
-5.5 (-6.7; -4.3)8 -4.3 (-6.0; -2.5) -5.9 (-6.4; -5.3)
1.6 (1.3; 1.8) 1.3 (1.0; 1.7) 1.6 (1.5; 1.7)
Nofe. HRBC-primed spleen cells from a pool of 3-month-old mice (10 animals) were filtered through two glass wool columns. After each passage, 1 x 106nucleated cells were immunized in vitro with 2 x 1oJTNP-HRBC while 5-40 x lo4 nucleated cells were titrated for in vifro helper cell activity (see Materials and Methods). The anti-TNP responses at Day 5 of culture showed that macrophage depletion after the second passage on glass wool was efficient in reducing the response of the carrierprimed cells (second column) but did not affect their helper cell activity as evidenced by invariance of the intercept and slope of the linear regression log PFCkulture on log primed cells (third and fourth columns). a 95% confidence limits in parentheses.
AGING
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FUNCTIONS
205
When spleen cells were immunized with a T-dependent antigen the anti-TNP antibody responsiveness declined after 3 months of age (Fig. 1). However, the different profiles of the anti-TNP response of spleen cells immunized with TNP-HRBC or TNP-Ficoll at the age of l-5 months indicate that alterations in the B-cell population are not sufficient to explain the age-dependent variation in the response to a T-dependent antigen. Thus, in this case, changes in the T-cell population could play a substantial role. Several studies have, indeed, shown that the decline of immune functions during senescence is due to changes in the T-cell compartment of the immune system (l-3,25,3 1,32) which have been attributed to thymic involution (33). Titration of helper cell activity revealed in the spleens of l- to lo-month-old mice the existence of two T-cell subpopulations (slope > 1) which interact to generate the helper activity of carrier-primed spleen cells (Fig. 6). The existence of a T cell-T cell cooperation in carrier-primed cell populations has been postulated by Janeway (34) to explain the helper cell “premium effect” observed during min vivo anti-hapten antibody response. A synergistic interaction between T-cell subpopulations with different Ly phenotypes has been suggested to play a role in the amplification of the antibody response (35,36). More recently, two helper cells have been identified by their different radiosensitivities (17) and different nylon adherence and antigenic (la) properties (37). In this last study, the two helper cells found in the spleens of adult mice and denoted Thl and Th2 were shown to be Ly l+ and to act synergistically by two independent mechanisms: Thl cells recognize the carrier moiety of the conjugate, Th2 cells provide polyclonal activation. The present finding of slope = 1 at 1l- 12 months of age indicates loss of the synergism between Thl and Th2 cells. In older mice the titration of helper cell activity revealed slopes < 1, suggesting the occurrence of suppressive events that counteract the activity of the helper cell population. The possibility that the concentration of suppressor cells is increased in old mice is supported by the results of several experiments (3-6, 11). However, it is difficult to reconcile an expansion of the suppressor T-cell population with thymic involution in aging (33). Characterization of antigen-specific suppressor T cells in aging mice should be instrumental in unraveling this problem. It should be pointed out that also for l- to lo-month-old mice the helper cell activity, as titrated in the present study, is probably the net result of interactions between helper and suppressor cells present in the carrier-primed cell population. Furthermore, Ly1,2,3+ cells in the normal cell population from 3-month-old mice might be activated by carrier-primed cells to amplify help or suppression (36). Therefore, the observed anti-TNP response is bound to reflect a balance of these regulatory events. As a matter of fact, it was found in preliminary experiments (data unpublished) that pretreatment of normal spleen cells from 3-month-old donors with mouse anti-Thy 1.2 antiserum and rabbit C enhanced the in vitro an&TNP antibody response to TNP-HRBC upon addition of HRBC-primed spleen cells from 3-month-old donors. This result is in line with the existence of feedback suppression circuits between primed and normal cells in the system used. In the present study it was found that the variability among mice of the same age in the in vitro antibody response to a T-dependent immunogen increases exponentially with aging (Fig. 1). This finding suggests that in aging mice the immune system is affected by impairing events of a stochastic nature. Impairing
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events of the same nature might also occur in other cell systems. Thus, accumulation of a crucial amount of these random events in some individuals of the same age may account for the variability in the onset and severity of senescence. In conclusion, events leading to impairment of immune responsiveness occur in old mice at multiple cellular levels. In addition to a marked defect in B-cell reactivity we have evidenced an age-related decrease in T-cell ability to interact synergistically in the generation of helper activity. It remains to be demonstrated whether this defect in T cell-T cell cooperation is intrinsic to the helper cell subpopulations or is the result of suppressor T-cell activity. REFERENCES 1. 2. 3. 4. 5.
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31,983,
1976. 6. Goidl, E. A., Innes, J. B., and Weksler, M. E., J. Exp. Med. 144, 1037, 1976.
7. Abraham, C., Tal, Y., and Gershon, H., Eur. J. Immunol. 7, 301, 1977. 8. Perkins, E. H., J. Reticuloendothel. Sot. 9, 642, 1971. 9. Heidrick, M. L., and Makinodan, T., J. Zmmunol. 111, 1502, 1973. 10. Callard, R. E., Eur. J. Immunol. 8, 697, 1978. 11. Doria, G., D’Agostaro, G., and Poretti, A., Immunology 35, 601, 1978. 12. Kettman, J., and Dutton, R. W., J. Immunol. 104, 1558, 1970. 13. Rittenberg, M. B., and Pratt, K. L., Proc. Sot. Exp. Biol. Med. 132, 575, 1969, 14. Doria, G., Mancini, C., and DeLisi, C., J. Zmmunol. 121, 2030, 1978. 15. Inman, J. K., J. Zmmunol. 114, 704, 1975. 16. Mishell, R. J., and Dutton, R. W., J. Exp. Med. 126, 423, 1967. 17. Agarossi, G., Pozzi, L., Mancini, C., and Doria, G., J. Zmmunol. 121, 2118, 1978. 18. Kettman, .I., and Dutton, R. W., Proc. Nat. Acad. Sci. USA 68, 699, 1971. 19. Mosier, D. E., and Coppleson, L. W., Proc. Nat. Acad. Sci. USA 61, 542, 1%8. 20. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. 21. Steel, R. G. D., and Tonic, J. H., ‘Principles and Procedures of Statistics.” McGraw-Hill, New York, 1960. 22. Edwards, A. A., and Dennis, J. A., “POLYFIT-A Computer Program for Fitting a Specified Degree of Polynomial to Data Points.” National Radiological Protection Board, Report NRPB-Mll. Harwell, U.K., 1973. 23. Albright, J. F., and Makinodan, T., J. Celi. Comp. Physiol. 67(Suppl. l), 185, 1%8. 24. Kishimoto, S.. Tsuyuchi, I., and Yamamura, Y., Clin. Exp. Immunol. 5, 525, 1969. 25. Kishimoto, S., and Yamamura, Y., Clin. Exp. fmmunol. 8, 957, 1971. 26. Kishimoto, S., Takahama, T., and Mizumachi, H., J. Immunol. 116, 294, 1976. 27. Segre, M. A., and Segre, D., J. Immunol. 116, 731, 1976. 28. Schneider, E. L., Fed. Proc. 38, 1857, 1979. 29. Gerbase-De Lima, M., Wilkinson, J., Smith, G. S., and Walford, R. L., J. Gerontol. 29,261, 1974. 30. Stutman, O., J. lmmunol. 109, 602, 1972. 31. Hori, Y., Perkins, E. H., and Halsall, M. K., Proc. Sot, Exp. Biol. Med. 144, 48, 1973. 32. Meredith, P., Gerbase-De Lima, M., and Walford, R. L., Exp. Gerontol. 10, 247, 1975. 33. Hirokawa, K., and Makinodan, T., J. Zmmunol. 114, 1659, 1975. 34. Janeway, C. A., J. Immunol. 114, 1394, 1975. 35. Feldmann, M., Beverley, P. C. L., Woody, J., and McKenzie, I. F. C., J. Exp. Med. 145,793,1977. 36. Tada, T., In “Immune System: Genetics and Regulation” (E. E. Sercarz, L. A. Herzenberg, and C. F. Fox, Eds.) pp. 345-361. Academic Press, New York, 1977. 37. Tada, T., Takemori, T., Okumura, K., Nonaka, M., and Tokuhisa, T., J. Exp. Med. 147,446,1978.
IMMUNOLOGY
53, 195-206 (1980)
Age-Dependent Changes of B-Cell Reactivity and T Cell-T Interaction in the in Vitro Antibody Response’ GINO DORIA, CNEN-Euratom
GIACOMO
D’AGOSTARO,
Cell
AND MAURO GARAVINI
Immunogenetics Group, Laboratory of Radiopathology, CSN Casaccia, Rome, Italy Received October 25, 1979
The in vitro anti-2,4,6-trinitrophenyl (TNP) antibody response of spleen cells from 1- to 32-month-old mice was evaluated by techniques suitable for dissecting out T- and B-cell reactivities. Spleen cells from individual mice carrier primed with horse red blood cells (HRBC) were immunized in vitro with TNP-HRBC, a T-dependent immunogen. The anti-TNP antibody response was found to change with the increasing age at which the animals were carrier primed, so that the response at 24 months was only about 4% of that at 3 months of age. It was also found that the variability in response among mice of the same age increased exponentially with age, suggesting that during senescence the immune system is impaired by stochastic events. A substantial part of the decline in immune responsiveness reflects the age-dependent decrease of B-cell reactivity as shown by marked reduction of the anti-TNP antibody response of spleen cells from old mice uponin vitro immunization with TNP-Ficoll, a T-independent immunogen. To analyze helper T-cell activity of aging mice independently from age-associated changes in B-cell reactivity the helper cell function was titrated by adding graded numbers of HRBC-primed spleen cells from individual mice of a given age to cultures containing a constant number of normal spleen cells from a pool of 3-month-old mice and the immunogen TNP-HRBC. The anti-TNP antibody response increases with the number of primed cells added to culture and the log number of anti-TNP plaque-forming cells is a linear function of the log number of the primed cells added. This linear regression was used to evaluate T cell-T ceil interactions. This analysis unmasked in aging mice a decrease in the ability of T cells to interact synergistically in the generation of helper activity.
INTRODUCTION In several animal species the antibody response increases during postnatal development, reaches a maximum in adulthood, and then declines during senescence. Changes in the population of immunologically competent cells and their functional consequences account for most of the variation of immunological competence (1). Thus, the decline of immune responsiveness in old mice is compatible with a decrease in the size and reactivity of the helper T-cell population (2, 3), an increase in the suppressor T-cell population (3-6), or both. Thymic involution, which is a prominent phenomenon during senescence, points to changes in T-cell populations as an important cause of the decline in immune functions. r This work was supported by a CNEN-Euratom Biology Division.
contract. Publication No. 1610 of the Euratom
195 0008-8749/80/090195-12$02.00/O Copyright Q 1980by AcademicPress,Inc. AU rights of reproduction in any form reserved.
1%
DORIA,
D’AGOSTARO,
AND GARAVINI
Alterations of the B-cell population have also been described in aged animals (1,3, 7). Macrophages seem to be unaffected by aging (8-10). We have previously described a parabolic rise and fall of the antibody response and affinity in aging mice, which have been attributed mainly to changes in type and degree of the cell interactions involved in the immune response (11). In the present study, a cellular analysis of age-dependent variations of the in vitro antibody response was carried out by techniques suitable to dissect out T- and B-cell reactivities. Functions of both T and B cells were found to change with aging. MATERIALS
AND METHODS
Animals and their carrierpriming. Male (C57BVlO x DBA/2) F, mice were used when 1-32 months old. For induction of helper cells, mice of a given age were carrier-primed by injecting iv 2 x lo5 (if not otherwise specified) horse red blood cells (HRBC)’ in 0.2 ml PBS 4 days before sacrifice. Red blood cells. HRBC and SRBC were prepared from blood suspended in Alsever’s solution, purchased from Sclavo (Siena, Italy), and washed repeatedly with PBS. TNP-RBC. TNP-HRBC were prepared by heavy coupling of TNBS to HRBC (12) and used as a T-dependent immunogen. TNP-SRBC were prepared by light coupling of TNBS to SRBC (13) and used as test antigen in the Jerne technique to detect direct anti-TNP PFC. These hemolytic plaques were shown to be TNP specific by inhibition with soluble TNP-bovine y-globulin or TNP-•-aminocaproic acid (14). TNP-Ficoll. TNBS was conjugated to Ficoll (MW 400,000) from Pharmacia (Uppsala, Sweden) according to Inman (15). The conjugate TNP-Ficoll with a molar ratio of 79: 1 was used as T-independent immunogen. Cell culture. Spleen cell suspensions were prepared from normal or carrierprimed mice and cultured by the method of Mishell and Dutton (16) using microtissue culture plates (Falcon No. 3040, Oxnard, Calif.) as previously described (17). Culture medium was Eagle’s minimum essential medium (Microbiological Associates, Bethesda, Md.) supplemented with 10% fetal calf serum (Rehatuin, Reheis, Phoenix, Ariz.), 1 miV sodium pyruvate, 0.1 m&f nonessential amino acid mixture, 100 IU/ml penicillin, 100 pg/ml streptomycin sulfate, 2 mkf L-glutamine (all from Microbiological Associates, Bethesda, Md.), and 20 m&f Hepes buffer solution (Eurobio, France). No mercaptoethanol was added to culture. This culture system was used to evaluate in vitro antibody responses to T-dependent and T-independent immunogens and to titrate in vitro helper cell activity, as described below. In vitro antibody response. The anti-TNP antibody response to a T-dependent or T-independent immunogen was elicited by immunizing spleen cells from carrierprimed or normal mice with TNP-HRBC or TNP-Ficoll, respectively. The immunization conditions were the following: 1 x lo6 nucleated spleen cells in 0.1 ml medium containing 2 x lo5 TNP-HRBC or 1.5 x 10” nucleated spleen cells in 0.2 ml medium containing 20, 2, or 0.2 ng TNP-Ficoll. After 4, 5, and 6 days of 2 Abbreviations used: HRBC, horse red blood cells; SRBC, sheep red blood cells; PBS, phosphatebuffered saline; TNP, 2,4,6&initrophenyl; TNBS, 2,4,6-trinitrobenzensulfonic acid; PFC, plaqueforming cells.
AGINGOFB-ANDT-CELLIMMUNEFUNCTIONS
197
culture the cells were harvested from four to eight wells of the same experimental group, pooled, washed, and assayed for direct anti-TNP PFC. The response was evaluated by the number of anti-TNP PFC per culture well. In the experiments with TNP-HRBC the number of PFC per spleen was calculated as PFC per culture x total nucleated cells per spleen. The results are presented as the peak responses found on Days 4 and 5 for TNP-Ficoll and TNP-HRBC, respectively. The T dependence or T independence of the response to the immunogens used was verified in preliminary experiments as follows. The anti-TNP antibody response of HRBC-primed spleen cells immunized with TNP-HRBC was shown to be impaired by pretreatment of the primed cells with mouse anti-Thy 1.2 antiserum and rabbit C. This cytotoxic treatment, however, did not affect the anti-TNP antibody response of normal spleen cells immunized with TNP-Ficoll. In each experiment with carrier-primed spleen cells and TNP-HRBC, the spleen cells were derived from 5 or 6 individual mice of a given age and from a pool of 3-month-old mice (5 animals). Since mice of different ages were tested in different experiments, the response of the spleen cells from each mouse was expressed as the percentage of the response of the pooled spleen cells used in the sameexperiment as reference age control. When normal spleen cells were immunized with TNP-Ficoll, pooled mice (8- 10 animals/group) of different ages were compared in the same experiment and the anti-TNP responses could be expressed directly as numbers of PFC per culture well. Titration of in vitro helper cell activity. A modification of the experimental model of Kettman and Dutton (18) was used to assay the level of helper cell activity in mice carrier primed at different ages. Normal spleen cells immunized in vitro with TNP-HRBC respond poorly to TNP. If graded numbers of spleen cells from HRBC primed mice are added to a constant number of normal spleen cells the anti-TNP response increases with the number of primed cells in the range where helper activity is limiting (17). If carrier-primed spleen cells from mice of different ages are added to normal spleen cells from mice of a constant age the evaluation of helper activity is independent from age-associated changes in B-cell reactivity which would affect in vivo estimates. In each experiment, all culture wells received 0.1 ml medium containing 2 x lo5 TNP-HRBC and 1 x IO6nucleated normal spleen cells from a pool of 3-month-old mice (lo- 15 animals) and were divided into three groups according to the addition of HRBC-primed cells. These cells were sampled from the same spleen suspensions also used in parallel cultures to determine the anti-TNP response as described in the preceding section. Culture wells of one group received no additional cells and served as baseline controls. Culture wells of the other two groups in addition received graded numbers (5-40 X 104) of nucleated spleen cells either from individual HRBC-primed mice of a given age or from a pool of HRBC-primed 3-month-old mice (5 animals) which served as reference age control. Cells from the same four to eight wells were harvested on Day 4,5, and 6 of culture and assayed for the number of direct anti-TNP PFC per culture well. The peak responses were found on Day 5 and processed as follows. The anti-TNP response of the normal spleen cells, which was very low (less than 10% of the response of 1 x lo6 nucleated spleen cells from a pool of carrier-primed 3-month-old mice), was subtracted from the responses of the normal spleen cells supplemented with primed cells. The net response was found to increase with the number of primed cells added to culture, so that the log number of
198
DORIA, D’AGOSTARO, AND GARAVINI
PFC was a linear function of the log number of primed cells added. Linear regressions were calculated for spleen cells from individual mice of a given age and for the pooled spleen cells from 3-month-old mice. The regression coefficient, which should represent the number of interacting cell populations (11, 19), was taken as an estimate of the degree of T cell-T cell interactions within the population of carrier-primed spleen cells that help the response of normal spleen cells. In control experiments, pooled spleen cell suspensions from carrier-primed 3-month-old mice were filtered through glass wool columns (20) before culture. Statistical analysis. Linear regressions and correlations were calculated as described by Steel and Torrie (21). Polynomial regressions were fitted by a previously described method (22). RESULTS Dose of Carrier Priming The optimal dose of HRBC to carrier-prime mice of different ages was determined in a preliminary experiment in which five mice at the ages of 1,3, or 20 months were injected with 2 x 104-2 x IO6HRBC. The animals were sacrificed 4 days later and pooled spleen cells from the carrier-primed mice of each group and from five unprimed age-control mice were immunized in vitro with 2 x IO5 TNP-HRBC. The results in Table 1 show that the anti-TNP peak response of spleen cells, expressed as PFC per culture or PFC per spleen, is greatly enhanced by carrier priming and that 2 x lo5 HRBC is the optimal priming dose at the three ages investigated. This dose was chosen for the subsequent experiments. TABLE 1 Effect of HRBC Priming on the in Vitro Anti-TNP Antibody Response of Spleen Cells from Aging Mice Age (months)
HRBC
PFCkulture
PFC/spleen
1
0 2 x 104 2 x 105 2 x log
16 110 248 193
1,352 9,626 32,552 21,203
3
0 2 x 104 2 x 105 2 x 106
26 553 888 508
4,762 99,440 157,318 107,925
20
0 2 x 104 2 x 105 2 x 106
14 13 511 108
2,116 1,706 66,021 13,594
Note. Spleen cells from mice unprimed or primed with different numbers of HRBC were cultured with 2 x 1W TNP-HRBC. Peak responses obtained at Day 5 of culture are referred to 1 X 106 nucleated cells initially put in culture. The number of anti-TNP PFCkulture was multiplied by the total number of nucleated cells/spleen to obtain the number of PFC/spleen.
AGING OF B- AND T-CELL IMMUNE FUNCTIONS
199
In Vitro Antibody Response Mice at the age of l-27 months were primed with 2 x lo5 HRBC and, 4 days later, individually tested for the ability of their spleen cells to mount an in vitro anti-TNP antibody response upon immunization with TNP-HRBC. The results of 14 experiments, in each of which five or six mice were individually tested, are reported in Fig. 1. The anti-TNP response of the spleen cells from each mouse is expressed as percentage of the response of the pooled spleen cells from 3-month-old mice used in the same experiment as reference age control. The upper part of Fig. 1 illustrates that after the initial rise from 1 to 3 months, the antibody response declines with increasing age so that at 24 months it is about 4% of that at 3 months.
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FIG. 1. In vitro anti-TNP antibody response of HRBC-primed spleen cells Born aging mice upon immunization with TNP-HRBC. (Top) Each point represents the response of spleen cells from a single mouse of a given age expressed as a percentage of the response of spleen cells from a pool of 3-month-old mice (reference age control). The number of PFC refers to 1 x 109nucleated cells initially put in culture. The line represents a second-order polynomial regression fitting all points including a zero value (omitted from the semilog diagram) of a 27-month-old mouse. (Bottom) Coefficient of variability: 100 x SD/ mean. The line represents the best-fitting regression calculated by the least-squares method.
200
DORIA,
D’AGOSTARO,
AND GARAVINI
Furthermore, this decline with age is concomitant with an increasing spread of the responses of individual mice. The lower part of Fig. 1, indeed, shows that the variability of the response increases exponentially with age, as evidenced by a statistically significant (P < 0.01) correlation (r = 0.82) between the log coefficient of variability and age. The decrease in antibody response of 1 x lo6 nucleated cells in culture could result from age-dependent alterations of the spleen cell populations such as diminution of the absolute number of immunologically competent cells or presence of abnormal (malignant) cells diluting out the number of competent cells in the sample tested in culture. The latter possibility might have occurred in 5 mice in which the range in total numbers of nucleated spleen cells was 2.9-9 x 108. However, the spleens of the other 66 mice contained 1-25 x lo8 nucleated cells, a range in cell numbers too narrow to account for the observed variation in antibody response. The possibility that the decline in response reflects an absolute decrease in the spleen pool size of immunologically competent cells is suggested by the demonstration in Fig. 2 that: (a) the response of 1 x lo6 nucleated spleen cells from all 71 mice is not correlated (r = 0.18;P > 0.05) with the total number of nucleated spleen cells (Fig. 2, right); (b) the response of 1 x lo6 nucleated spleen cells not only is correlated (r = 0.75; P < 0.01) with the response per spleen but, if the most extreme point (highest number of PFUspleen) is disregarded, the linear regression coefficient f SE is 0.93 + 0.04, avalue very close to 1 as expected if the spleen size
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201
AGING OF B- AND T-CELL IMMUNE FUNCTIONS
does not affect the in vitro response (Fig. 2, left). However, it cannot be excluded that the decline in response reflects qualitative rather than quantitative abnormalities of immunologically competent cells or a combination of the two possibilities. The age-dependent decline of the in vitro antibody response of carrier-primed spleen cells to a T-dependent antigen such as TNP-HRBC is compatible with alterations of the T- and B-cell populations. Defects in the B-cell population were demonstrated by the finding of a decrease in the anti-TNP response of normal spleen cells from old mice immunized in vitro with TNP-Ficoll, a T-independent antigen. The results of the experiment reported in Fig. 3 describe an increase of the response from 1 to 5 months of age followed by a continuous decrease until 32 months. This parabolic variation of the response with aging was consistent at three antigen doses. Titration of In Vitro Helper Cell Activity The helper function of T cells was titrated by adding graded numbers of carrier-primed spleen cells from individual mice of a given age to cultures of a constant number of normal spleen cells from a pool of 3-month-old mice. Upon immunization with TNP-HRBC, the in vitro anti-TNP antibody response of the normal spleen cells increased with the number of added carrier-primed spleen cells. Figure 4 describes the results of an experiment in which five individual mice were tested at the age of 3 months. When the linear regressions (log PFCkulture on log primed cells added to culture for individual mice) are compared to the regression for the reference age control (carrier-primed spleen cells from a pool of five mice at the age of 3 months) the straight lines for individual mice overlap to a great extent with the control line as expected, although some individual variation is also evident. The
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202
DORIA, D’AGOSTARO, AND GARAVINI AGE: 3 -
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FIG. 4. Titration of in vitro helper cell activity by the linear regression log PFCkulture on log primed spleen cells added to cultures of normal spleen cells from a pool of 3-month-old mice. Straight lines for primed spleen cells from five mice individually tested at 3 months are compared to the line (dashed) for the primed spleen cells from a pool of five mice tested at 3 months (reference age control). The lines represent best-fitting regressions calculated by the least-squares method. Slopes are represented by b values.
results of another experiment reported in Fig. 5 illustrate that the slopes of the straight lines for five individual mice tested at the age of 24 months are always lower than those for the reference age control, indicating a striking defect in helper cell activity. Linear regressions (log PFC/culture on log primed cells) were calculated for 71 mice individually tested at different ages in 14 experiments as well as for the pool of 3-month-old mice used in each experiment as reference age control. The mean slope 4 SE of the linear regressions for the reference age controls from all experiments is 1.38 ? 0.08. Since the standard error is very small (6%) the regression coefficients for individual mice tested in different experiments are reported in Fig. 6 without normalization to the reference age controls. The data indicate age-dependent alterations in type and degree of T cell-T cell interaction within the carrier-primed spleen cell population. Positive cooperation between two T-cell subpopulations is evident at l- 10 months (coefficient > l), negligible at 1I- 12 months (coefficient = I), and inhibited at 15, 18, 21, 24, and 27 months (coefficient < 1). The interpretation of the titration data on helper cell activity is based on the assumption that B cells and macrophages of the carrier-primed cell population do not contribute to the anti-TNP response of normal spleen cells. The irrelevance of
203
AGING OF B- AND T-CELL IMMUNE FUNCTIONS AGE:
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FIG. 5. Titration of in virro helper cell activity by the linear regression log PFCkulture on log primed spleen cells added to cultures of normal spleen cells from a pool of 3-month-old mice. Straight lines for primed spleen cells from five mice individually tested at 24 months are compared to the line (dashed) for the primed spleen cells from a pool of five mice tested at 3 months (reference age control). The lines represent best-fitting regressions calculated by the least-squares method. Slopes are represented by b values.
the B-cell component of the primed cells was demonstrated in previous experiments (17) by the same anti-TNP response of 1 X lo6 normal spleen cells cultured with 30 x lo4 HRBC-primed spleen cells untreated or pretreated with rabbit anti-mouse Ig antiserum and guinea pig C, a treatment shown to be very effective in abrogating the in vitro primary response of normal spleen cells to SRBC. The contribution of macrophages was ruled out by the demonstration that efficient removal of macrophages from the carrier-primed spleen cell population by two passages through glass wool does not affect helper cell activity (Table 2). DISCUSSION The analysis of T- and B-cell reactivities as carried out in the present study revealed new and marked alterations in the lymphocyte populations of aging mice, implementing and clarifying previous results from in vivo and in vitro studies on the senescence of the immune system (l-3,9,11,23-27). The changes in T- and B-cell reactivities as evidenced by the present analysis might have resulted from age-dependent alterations in cell replication capacity arising in vitro rather than preexisting in vivo . However, since the same changes in percentage replicating cells and cell population doubling time have been shown to occur both in vitro and in vivo
204
DORIA, D’AGOSTARO, AND GARAVINI
T 0 1
T 0 1
2
FIG. 6. Changes in T cell-T cell interactions with aging. Each point represents the mean slope of linear regressions log PFClculture on log primed spleen cells from 5 to 10mice individually titrated for in virro helper cell activity. Vertical bars denote -C1SE. The dashed line is positioned at 1 which is the expected slope value when no interaction occurs within the carrier-primed spleen cell population.
(28), the decline of in vitro immune functions with increasing age can be attributed to in vivo cell alterations. The anti-TNP antibody responsiveness of spleen cells immunized with a T-independent antigen was found markedly decreased in old mice. Defects in the B-cell population appear after 5 months of age as evidenced by a IO-fold reduction in the antibody response to TNP-Ficoll (Fig. 3). The extent of impairment of this response is at variance with the only slight decrease observed in the mitotic responsiveness of B lymphocytes to lipopolysaccharide (1, 26, 29) and with the finding that the population size of B lymphocytes, as detected by surface markers, is not decreased in old mice (30). Thus, qualitative abnormalities in the B-cell population seem to play a greater role than cell depletion (3). TABLE 2 Persistent Helper Cell Activity of Carrier-Primed Spleen Cells after Macrophage Depletion Log PFC vs log primed cells regression Passage on glass wool None 1 2
PFCkulture 875 825 163
Intercept
Slope
-5.5 (-6.7; -4.3)8 -4.3 (-6.0; -2.5) -5.9 (-6.4; -5.3)
1.6 (1.3; 1.8) 1.3 (1.0; 1.7) 1.6 (1.5; 1.7)
Nofe. HRBC-primed spleen cells from a pool of 3-month-old mice (10 animals) were filtered through two glass wool columns. After each passage, 1 x 106nucleated cells were immunized in vitro with 2 x 1oJTNP-HRBC while 5-40 x lo4 nucleated cells were titrated for in vifro helper cell activity (see Materials and Methods). The anti-TNP responses at Day 5 of culture showed that macrophage depletion after the second passage on glass wool was efficient in reducing the response of the carrierprimed cells (second column) but did not affect their helper cell activity as evidenced by invariance of the intercept and slope of the linear regression log PFCkulture on log primed cells (third and fourth columns). a 95% confidence limits in parentheses.
AGING
OF B- AND T-CELL
IMMUNE
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When spleen cells were immunized with a T-dependent antigen the anti-TNP antibody responsiveness declined after 3 months of age (Fig. 1). However, the different profiles of the anti-TNP response of spleen cells immunized with TNP-HRBC or TNP-Ficoll at the age of l-5 months indicate that alterations in the B-cell population are not sufficient to explain the age-dependent variation in the response to a T-dependent antigen. Thus, in this case, changes in the T-cell population could play a substantial role. Several studies have, indeed, shown that the decline of immune functions during senescence is due to changes in the T-cell compartment of the immune system (l-3,25,3 1,32) which have been attributed to thymic involution (33). Titration of helper cell activity revealed in the spleens of l- to lo-month-old mice the existence of two T-cell subpopulations (slope > 1) which interact to generate the helper activity of carrier-primed spleen cells (Fig. 6). The existence of a T cell-T cell cooperation in carrier-primed cell populations has been postulated by Janeway (34) to explain the helper cell “premium effect” observed during min vivo anti-hapten antibody response. A synergistic interaction between T-cell subpopulations with different Ly phenotypes has been suggested to play a role in the amplification of the antibody response (35,36). More recently, two helper cells have been identified by their different radiosensitivities (17) and different nylon adherence and antigenic (la) properties (37). In this last study, the two helper cells found in the spleens of adult mice and denoted Thl and Th2 were shown to be Ly l+ and to act synergistically by two independent mechanisms: Thl cells recognize the carrier moiety of the conjugate, Th2 cells provide polyclonal activation. The present finding of slope = 1 at 1l- 12 months of age indicates loss of the synergism between Thl and Th2 cells. In older mice the titration of helper cell activity revealed slopes < 1, suggesting the occurrence of suppressive events that counteract the activity of the helper cell population. The possibility that the concentration of suppressor cells is increased in old mice is supported by the results of several experiments (3-6, 11). However, it is difficult to reconcile an expansion of the suppressor T-cell population with thymic involution in aging (33). Characterization of antigen-specific suppressor T cells in aging mice should be instrumental in unraveling this problem. It should be pointed out that also for l- to lo-month-old mice the helper cell activity, as titrated in the present study, is probably the net result of interactions between helper and suppressor cells present in the carrier-primed cell population. Furthermore, Ly1,2,3+ cells in the normal cell population from 3-month-old mice might be activated by carrier-primed cells to amplify help or suppression (36). Therefore, the observed anti-TNP response is bound to reflect a balance of these regulatory events. As a matter of fact, it was found in preliminary experiments (data unpublished) that pretreatment of normal spleen cells from 3-month-old donors with mouse anti-Thy 1.2 antiserum and rabbit C enhanced the in vitro an&TNP antibody response to TNP-HRBC upon addition of HRBC-primed spleen cells from 3-month-old donors. This result is in line with the existence of feedback suppression circuits between primed and normal cells in the system used. In the present study it was found that the variability among mice of the same age in the in vitro antibody response to a T-dependent immunogen increases exponentially with aging (Fig. 1). This finding suggests that in aging mice the immune system is affected by impairing events of a stochastic nature. Impairing
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events of the same nature might also occur in other cell systems. Thus, accumulation of a crucial amount of these random events in some individuals of the same age may account for the variability in the onset and severity of senescence. In conclusion, events leading to impairment of immune responsiveness occur in old mice at multiple cellular levels. In addition to a marked defect in B-cell reactivity we have evidenced an age-related decrease in T-cell ability to interact synergistically in the generation of helper activity. It remains to be demonstrated whether this defect in T cell-T cell cooperation is intrinsic to the helper cell subpopulations or is the result of suppressor T-cell activity. REFERENCES 1. 2. 3. 4. 5.
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