6 mice

CELLULAR

IMMUNOLOGY

63, 16-27 (1981)

The Immune Response

in Aged C57BL/6

Mice

I. Assessment of Lesions in the B-Cell and T-Cell Compartments of Aged Mice Utilizing the Fc Fragment-Mediated Polyclonal Antibody Response’ EDWARD L. MORGAN,**’ MARILYN L. THOMAN,~ AND WILLIAM 0. WEIGLE Department of Immunopathology. Scripps Clinic and Research Foundation, La Jolla. California 92037 Received January 20. 1981; accepted April 30, 1981 The Fc fragment-mediated polyclonal antibody response was utilized to assess B-cell, Tcell, and macrophage reactivity in aged C57BL/6 mice. Spleen cells from aged (28-30 months) mice were found to be deficient in their capacity to proliferate and produce polyclonal antibody in response to Fc fragments when compared to adult (2-3 months) controls. Since T cells are required for the Fc-induced polyclonal antibody response,T cells from aged mice were assessed for their ability to restore the polyclonal antibody response in T-cell-depleted adult spleen cell populations. Aged T cells were not as effective as adult T cells in restoring the antibody response. The T-cell requirement in the Fc-induced polyclonal response has been shown to be replaceable by the Fc-stimulated T-cell replacing factor (Fc)TRF. T cells derived from aged mice were unable to produce (Fc)TRF to the level of adult cells. In addition to a defect in the T-cell compartment a lesion exists in the B-cell compartment of aged mice as well. Adult T cells were not capable of restoring the polyclonal antibody response of aged B cells any higher than aged T cells indicating a B-cell defect. Moreover, when a direct B-cell activator, Fc subfragment, was employed, the aged B cells were not stimulated to the level of adult controls. To test the ability of aged macrophages to function as accessorycells in the polyclonal response,macrophage-depleted adult spleen cells were mixed with aged or adult macrophages and the responsemeasured. The results indicate that aged macrophages restore the polyclonal antibody response as efficiently as their adult counterpart.

INTRODUCTION The study of immune potential during senescencehas been the focus of numerous investigations in which it has been established that the magnitude of various parameters of the immune responsesdeclines with age. This decline is manifested by impaired capacity to develop both cell-mediated (l-3) and humoral immunity (414). Changes in both the thymus-derived (T) cell and bone-marrow-derived (B) ’ This is Publication No. 2304 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, Calif. This work was supported in part by United States Public Health Service Grants A107007, A115761 and AG00783, and Biomedical Research Support Program Grant RRO5514. ’ Recipient of United States Public Health Postdoctoral Fellowship AI058 13. 3To whom all correspondence should be directed. 4 Recipient of United States Public Health Service Postdoctoral Fellowship AI06085 16 0008-8749/81/130016-12$02.00/O Copyright 8 1981 by Academic Rosa. Inc. All rights of reproduction in any form reserved.

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RESPONSE IN AGED C57BL/6 MICE

17

cell compartments have been associated with the reduced ability of aged mice to respond to antigenic stimulation. Despite this general decay of immune function, increased age is also associated with the development of autoimmune disease states in both man (15) and the mouse (16). Thus, it is not surprising that decreased suppressor-T-cell activity has been reported to accompany the aging process ( 16). Little information is available on the parameters of the immune regulatory circuits, and few studies have been made using one system to simultaneously evaluate the effects of aging on different events involved in lymphocyte activation. As the result of recent studies on B-cell activation with Fc fragments of human immunoglobulin (Ig),5 it became obvious that this model would lend itself to such an approach, since Fc fragment-induced B-cell activation requires the complex interaction of multiple cell types. The Fc portion of Ig has the capacity to induce murine B cells to proliferate ( 17- 19) and produce polyclonal antibody (20). The cellular and molecular requirements have been extensively investigated and it has been established that both macrophage (18-20) and T cells (20) are required to drive B cells to polyclonal antibody production, whereas only macrophages are required for Fc fragment-mediated B-cell proliferation. Moreover, Fc fragments have been shown to possessadjuvant properties (21, 22). Fc fragments are able to potentiate both humoral (21-23) and cell-mediated (24) immune responses. Taken together these results indicate that the Fc fragment is a potent in vivo and in vitro immune regulator involving B cells, T cells, and macrophages. The purpose of this study was to evaluate the cellular parameters of Fc fragment-mediated activation in aged mice. MATERIALS

AND METHODS

Animals. Male mice of the inbred C57BL/6 strain age 2-3 months were obtained from Jackson Laboratories (Bar Harbor, Maine) and Charles River (Willmington, Mass.). Aged male C57BL/6 mice (28-30 months) were obtained from Charles River. All experiments were performed with adult mice from both sources with comparable results. Preparation of Fc fragments. A human IgG, myeloma protein (Fi) was a gift from Dr. Hans L. Spiegelberg, Scripps Clinic and Research Foundation. The IgG, was purified by ammonium sulfate fractionation followed by DEAE cellulose chromatography with 0.01 M phosphate buffer, pH 8, used as the eluent. Fc fragments were obtained by digestion of IgG, with papain (Sigma Chemical Co., St. Louis, MO.) in the presence of L-cysteine (Sigma) and ethylenediaminetetraacetic acid (EDTA) (J. T. Baker Chemical Co., Phillipsburg, N.J.) for 5 hr (25). Following digestion the material was chromatographed on Sephadex G-100 (Pharmacia Fine Chemicals, Piscataway, N.J.) to remove any undigested IgG. The Fc and Fab fragments were then separated from each other by DEAE chromatography (26). Preparation of Fc subfragments. Stimulatory adherent cell supernate was prepared as previously described ( 19). A single-cell suspension was prepared by teasing apart the spleens into cold phosphate-buffered saline (PBS). The suspension was allowed to stand for 5 min in ice to allow clumps to settle out. The cells were ’ Abbreviations used: FCS, fetal calf serum; Ig, immunoglobulin; PFC, plaque-forming cells; SRBC, sheep red blood cells; TNP, trinitrophenyl; TRF, T-cell replacing factor; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; S-III, pneumococcal polysaccharide type III.

18

MORGAN,

THOMAN,

AND

WEIGLE

washed twice and resuspended to a concentration of 2 X lO’/ml in RPM1 1640 (Flow Laboratories, Rockville, Md.), supplemented with 2 mM L-glutamine, 1% BME vitamins (Grand Island Biological Co., Grand Island, N.Y.), 100 units penicillin and 100 pg streptomycin (Microbiological Associates, Bethesda, Md.), 5 X lop5 A4 2-mercaptoethanol (2-ME), and 5% fetal calf serum (FCS) (Gibco). One-milliliter samples were allowed to adhere to tissue culture dishes (3001, Falcon Plastics, Oxnard, Calif.) for 1 hr in an atmosphere of 5% CO* at 37°C. The cells were washed extensively with RPM1 1640 to remove nonadherent cells prior to the addition of Fc fragments. One milligram of Fc in 2 ml RPM1 1640, supplemented with 2 mM L-glutamine, 1% vitamins, 100 units penicillin, 100 rg streptomycin, 5 X lop5 M 2-ME, and 0.5% fresh normal mouse serum, was added to the adherent cell layer. The supernatant material was collected after a 1-hr incubation period and centrifuged at 1500 g for 10 min in a refrigerated centrifuge and sterilized by filtration through at 0.22-pm filter prior to chromatographic separation. The adherent cell supernate was chromatographed on a Sephadex G-50 superfine (Pharmacia, Piscataway, N.J.) column (1.2 X 30 cm) with a bed volume of 20 ml. The material was eluted with PBS at a flow rate of 30 ml/hr and 0.5-ml samples were collected. The column was calibrated with the following standards, blue dextran (50,000 MW) (Pharmacia), deoxyribonuclease (3 1,000 MW) (Sigma), and lysozome (14,300 MW) (Calbiochem-Behring Corp., La Jolla, Calif.). The molecular weight of the mitogenic material was calculated from a plot of the elution volumes versus the logarithm of the molecular weights of the standards. Mitogenic activity was found to have a molecular weight of approximately 14,000 (19). The columnpurified material was then affinity purified on an anti-Fc affinity column (19) prior to use. Fc subfragment directly induces B cells to proliferate in the absence of accessory macrophages or T cells. Isolation of macrophages. Splenic macrophages were isolated as previously described (18, 19). Spleen cell preparations were subjected to 2000 R of irradiation from a Gamma Cell 40 small animal irradiator (Atomic Energy of Canada Ltd., Ottawa, Canada) delivered at 107 R/min prior to adherence. The irradiated spleen cells were adjusted to 6 X 10’ cells/O.5 ml RPM1 1640 + 10% FCS and 0.5 ml was allowed to adhere to the wells of microtiter plates (3040 Microtest II, Falcon) for 30 min in an atmosphere of 5% COZ at 37°C. The wells were washed extensively with RPM1 1640 to remove any nonadherent cells. These cells were used as a source of macrophages in restoration of the polyclonal response in macrophagedepleted cultures. Depletion of macrophages by Sephadex G-l Ofiltration. Spleen cells were filtered through columns of Sephadex G-10 (Pharmacia) by the method of Ly and Mishell (27). Briefly, 5 X 10’ spleen cells in 2 ml RPM1 1640 supplemented with 5% FCS were filtered through a column containing 9 ml of Sephadex G-10 which was previously equilibrated with RPM1 1640. The number of residual macrophages present was monitored by esterase straining (28). Sephadex G-10 passage reduced the esterase-positive cells from approximately 7- 10% to to. 1%. Depletion of T lymphocytes. T-Cell-depleted spleen cell populations were prepared as previously described ( 17). Rabbit anti-thymocyte serum (Microbiological Associates) was absorbed with a myeloma cell line (XS-63). Spleen cells were incubated at a concentration of 5 X lO’/ml in PBS with a 1:40 dilution of guinea pig complement (Pel-Freez, Inc., Rogers, Ark.) and incubated for 30 min at 37°C.

IMMUNE

RESPONSE

IN AGED

C57BL/6

MICE

19

The complement was preabsorbed for 30 min at 4°C with 1:5 packed volume of red blood cells and then with a mixture of thymocytes and spleen cells from C3H mice. Enrichment of T lymphocytes. The spleen cell population was enriched for T lymphocytes by filtration through nylon wool columns (Fenwal Laboratories, Deerfield, Ill.) (29). Production of Fc fragment-induced T-cell replacing factors. Spleen cells were incubated 24 hr in 0.2 ml RPM1 1640 supplemented with 2 mM L-glutamine, 100 units penicillin, 100 pg streptomycin, 5 X low5 M 2-mercaptoethanol, 5% fetal calf serum (FCS) (Grand Island Biological), and 50 pg/ml Fc fragments. Culture supernatants were collected and tested (lo-20 pi/well) for T-cell replacing activity in the Fc-induced polyclonal response.The culture supernatants were then subjected to Sephadex G-100 separation as described previously (30). Active fractions were pooled and stored at -20°C prior to use. Polyclonal antibody response assay. For the generation of the polyclonal plaqueforming-cell (PFC) response spleen cells were suspended to a concentration of 6 X 106/ml in RPM1 1640 supplemented with 2 mM glutamine, vitamins, 100 units penicillin, 100 pg streptomycin, 7.5% FCS, and 0.5% fresh normal mouse serum. Duplicate cultures of 6 X lo5 cells/O.3 ml were incubated in microtiter plates (3040 Microtest II, Falcon) at 37°C in 5% COZ. Duplicate cultures were harvested on Day 3 and assayed for a response to 2,4,6-trinitrophenyl (TNP) by the slide modification of the Jerne and Nordin plaque assay (3 1). Heavily TNP-conjugated sheep red blood cells (TNP-SRBC) were prepared according to the method of Kettman and Dutton (32) and were used as the indicator RBC. Guinea pig serum (PelFreez) was the source of complement to develop the direct or IgM plaques. Results of the plaque forming assay are expressed as the mean PFC/106 original cells of duplicate pools f SE. Each experiment was performed a minimum of three times and the experiments shown are representative of all the data. Proliferative response assay. The Fc fragment-mediated proliferative response was performed as previously described ( 17-19). RESULTS Fc Fragment-Induced

Activation

of Aged B Cells

To determine whether splenic B cells from aged (28-30 months) mice could be activated by the Fc portion of human Ig, they were compared to normal adult (3 months) B cells. The results in Fig. 1 demonstrated that Fc fragment-induced Bcell proliferation is greater in adult than aged spleen cell cultures. The low response of aged cells was not due to an increased stimulation threshold because the responses obtained with 50 and 100 pg were not significantly different. The optimal concentration of Fc for the adult proliferative response was found to be 100 pg/culture as described previously ( 18). Since B-cell proliferation is an integral part of Fc fragment-mediated polyclonal antibody production (20), it was important to determine whether aged spleen cell cultures also showed a reduced polyclonal antibody response. The results in Fig. 2 indicate that adult spleen cells produce a significantly greater polyclonal antibody responsecompared to aged spleen cells. As observed with the proliferative response, the optimal concentration of Fc needed for the polyclonal antibody response was

20

MORGAN,

THOMAN,

“‘1

AND

WEIGLE

nAged

T

80 60 40 20

Fc(pglCulture) FIG. 1. Comparison of the ability of aged and adult spleen cells to proliferate fragments. The response was measured on Day 3 of culture.

in response to Fc

100 pg/culture. The inability of aged spleen cells to produce polyclonal antibody to the level of adult controls was not due to a shift in the response kinetics because the low but significant antibody response produced by aged spleen cells peaked on Day 3 of culture (Fig. 3) as did the much larger response of the adult cells. Analysis of T- and B-Cell Reactivity in Aged Mice Since T cells are essential in the Fc fragment-induced polyclonal antibody response (20), it was important to ascertain whether the T cell was a defective component in aged mice. T cells from both aged and adult mice were assessedfor their ability to restore the polyclonal antibody response in T-cell-depleted adult spleen cell populations. The results in Fig. 4 show that aged T cells were not as effective as adult T cells in restoring the polyclonal antibody response. This ob-

3009

0 Aged

T

y 250b 5 200k F 150.& z looz E a 50-

L lb 5.0 lb0 Fc(pglCulture) FIG. 2. Comparison of the ability of aged and adult spleen cells to produce polyclonal response to Fc fragments. The response was measured on Day 3 of culture.

antibody

in

IMMUNE

RESPONSE IN AGED C57BL/6 MICE

300

1

o- -0 Aged -

y 250-

b 5 200E g 150c.L I loo-

!ii

d

21

Adult

A /--F..-

50

r--

--‘-3

1

7-T-

Dayof Assay

4

FIG. 3. Kinetic profile of the Fc fragment-induced polyclonal antibody response. 100 pg Fe/culture was used.

servation was found to be true over a wide range of B:T cell ratios. With both aged and adult T cells the optimal response was obtained with approximately equal numbers of T cells and B cells. The response obtained was comparable to that seen with untreated adult spleen cell preparations. The T-cell requirement in the polyclonal antibody response has recently been shown to be replaceable by supernatant material derived from T cells cultured with Fc fragments (30). This T-cell replacing factor (Fc)TRF, was found, in conjunction with Fc fragments, to stimulate T-cell-depleted cultures to make polyclonal antibody. T cells derived from aged mice were unable to produce (Fc)TRF to the level of adult cells (Fig. 5). The maximal amount of (Fc)TRF was generated at a cell

250 % 2200 Z :: y50

-Adult **Aged

T Cells T Cells

I-

E .A I100 t; .-e 0 50

% B Cells 100

75

50

25 I

4

25

50

75

100

I

% T Cells 0

0

FIG. 4. Comparison of the ability of aged and adult T cells to restore the Fc-mediated polyclonal antibody response in T-cell-depleted adult spleen cell cultures. Varying numbers of B and T cells were added together such that the final cell concentration was held constant at 6 X 10’ cells/culture. 100 rg Fe/culture was used and the response measured on Day 3.

22

MORGAN, THOMAN,

AND WEIGLE

20

&15 G k ;10 .& 2 t; 2 5 ii 5~10'1~10'5~10'1~10‘

(Fc)TRFGenerator Cells FIG. 5. Comparison of the ability of aged (0) and adult (a) spleen cells to produce (Fc)TRF. Increasing numbers of spleen ceils were incubated with Fc fragments (50 fig/ml) for 24 hr and the supernatants harvested. The supernatant material was subjected to G-100 chromatography prior to use. 20 rl/well of (Fc)TRF was added to T-cell-depleted adult spleen cell cultures along with 100 rg Fe/culture and the polyclonal antibody response measured on Day 3.

density of 5 X 104/0.2 ml for both adult and aged cultures. Taken together these results indicate that T cells from aged mice are not as efficient as adult T cells in supporting Fc fragment-induced polyclonal antibody production. To assesswhether a lesion existed in the B-cell compartment of aged mice, T cells from adult and aged mice were added to T-cell-depleted aged spleen cell populations and the Fc-mediated response was measured. The data expressed in Fig. 6 indicate that aged B cells as well as T cells are defective in the Fc polyclonal

1o--0ApdTcIel

400

HAMTCdlS

% B Cells 100

75

50

25

K T Cells 0

25

50

75 100

0

FIG. 6. Comparison of the ability of aged and adult T cells to restore the Fc-mediated polyclonal antibody response of T-cell-depleted aged spleen cell cultures. Varying numbers of B and T cells were added together such that the cell concentration was held constant at 6 X l@/culture. 100 rg Fe/culture was used and the response measured on Day 3.

IMMUNE

23

RESPONSE IN AGED C57BL/6 MICE TABLE 1

Comparison of the Ability of Fc Subfragments to Induce Aged and Adult Spleen Cells to Proliferate Spleen cells”

Fc subfragmen?

[‘H]TdR uptake culture -t SE’

Aged

+

2,085 + 706 7,765 + 1,127

Adult

+

1,587 f 378 34,387 k 3,361

a Spleen cells were filtered through Sephadex G-10 columns prior to stimulation with Fc subfragment. b 1.25 pgfculture. ’ The response was measured on Day 3 of culture.

response. Adult T cells were not capable of restoring the polyclonal antibody response any higher than aged T cells indicating that aged B cells were incapable of being stimulated to the level of adult B cells. Moreover, a shift in the optimal B:T cell ratio had not occurred since approximately equal numbers of B and T cells still produced the maximum response. To further evaluate the aged B cell, advantage was taken of the fact that Fc subfragments, derived from macrophage processing of Fc fragments, directly stimulate B-cell proliferation in the absence of accessory cells (19). When Fc subfragments were added to aged and adult spleen cell cultures, it was found that adult cells proliferated to a greater extent than aged spleen cells directly indicating a lesion in the B-cell compartment of aged mice (Table 1). Analysis of Macrophage Reactivity in Aged Mice To test the ability of aged macrophages to function as accessory cells in the Fcinduced polyclonal antibody response, macrophage-depleted adult spleen cells were added to irradiated adherent cell monolayers from aged and adult mice and the response was measured. The results in Fig. 7 reveal that aged macrophages restore the polyclonal antibody response as well as adult splenic macrophages. These results indicate that splenic macrophages from aged mice are as effective as adult macrophages in processing Fc fragments into mitogenic Fc subfragments, the previously reported function of macrophages in this polyclonal response. Sephadex G-10 Filtered Adult Cells

+ + +

Adult M.

+

Aged M,

+

+ +

Fc

+ + + + + 50 100 Direct Anti-TNPPFCllO”iSE

150

FIG. 7. Comparison of the ability of aged and adult splenic macrophages to restore the Fc-mediated polyclonal antibody response in macrophage depleted adult spleen cell cultures. 6 X 10’ Sephadex G-10 filtered spleen cells were added to microwells containing either adult or aged macrophages. 100 pg Fc/ culture was used and the response measured on Day 3 of culture.

MORGAN, THOMAN,

AND WEIGLE

TABLE 2 Inability of Aged T Cells to Suppress the Fc Fragment-Mediated Polyclonal Antibody Response by Adult Spleen Cells Adult spleen cells 6X 10’

Aged” T cells -

Fc fragments6

Direct anti-TNP PFC/106 f SE’

+

33k 1 295 f 35

+

16+ 3 300 k 27

+

22f 1 304 f 10

+

29k 308 +

1 x IO’ 1 x lo5 2 x lo5

4 5

’ Nylon-wool-nonadherent spleen cells. * 100 pg Fe/culture. c The responsewas measured on Day 3 of culture. The polyclonal antibody responseof untreated aged spleen cells was 96 f 7 PFC/106.

Analysis of Suppressor-T-Cell Activity in Aged Mice To assesswhether T cells derived from aged mice had the ability to suppress the Fc fragment-mediated polyclonal antibody response, increasing numbers of aged T cells were added to adult spleen cells and the response was measured. The results in Table 2 reveal that the aged T cells were unable to suppress the Fc-mediated polyclonal antibody response. Moreover, when adult T cells were substituted for the aged T cells, similar results were observed (data not shown). Untreated spleen cells from the aged mice were defective in their response to Fc when compared to adult controls (97 PFC vs 295 PFC). These results indicate that the inability of spleen cells from aged mice to respond to the levels of adult controls was not due to suppressor T cells. DISCUSSION The Fc portion of immunoglobulin serves as an ideal activator in the evaluation of lymphocyte and accessory cell reactivity in an aging model because both T cells (20, 30) and macrophages (20) are required to generate the polyclonal antibody response and macrophages are required for B-cell proliferation ( 18, 19). Both the proliferative and polyclonal antibody responsesto Fc fragments were reduced during the process of aging. Spleen cells from individual aged (28-30 months old) mice proliferated and produced polyclonal antibody at a significantly lower level compared to spleen cells from adult (3-month-old) control mice. Spleen cell preparations from aged mice were not pooled due to the observation that a large degree of variability exists in the magnitude of antibody responses from aged mice (33). Moreover, adult control spleen cell populations were not pooled for purposes of consistency. All experiments were performed with adults from both Charles River and Jackson with no observable differences. The data shown were obtained with adult mice from Charles River. Collectively, we have assessedthe polyclonal an-

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25

tibody responses of approximately 25 aged mice and all were significantly lower (average five-fold lower) than the adult control values. The reduced responsiveness was not due to a shift in the dose-response or kinetic profiles of the aged mice since the maximum responses obtained with aged spleen cells coincided with maximum responsesof adult controls. Callard et al. (6) employing bacterial lipopolysaccharide (LPS) and pneumococcal polysaccharide type III (S-III) also observed that the reduced level of polyclonal B-cell activity could not be explained in terms of alterations in the kinetics of the responding cells as the optimal dose and the time of the peak response for both LPS and S-III were similar for aged and adult cells. Moreover, these authors (6) showed that the defect was not due to a decrease in the number of B cells present because an increase rather than a decrease in B cells was found in spleen from aged mice. Although macrophages are required for the generation of both proliferation and polyclonal antibody production, they appear not to be affected in these capacities by the aging process. Splenic macrophages derived from aged mice are as efficient as adult macrophages in supporting the Fc fragment-induced polyclonal antibody response. It has previously been shown that the role of the macrophage in Fcinduced B-cell activation is the generation of mitogenic 14,000 MW Fc subfragments which then directly induce B cells to proliferate (18, 20). The addition of aged macrophages to macrophage-depleted adult spleen cells was found to restore the polyclonal antibody response. This observation is reminiscent of the work of Heidrick and Makinodan (8) who found that adherent cells from aged mice cultured with adult nonadherent cells responded fully to SRBC, whereas aged nonadherent cells combined with adult adherent cells gave a depressed response comparable to unseparated aged spleen cells. In contrast Nordin and Buchholz (9) demonstrated that peritoneally derived macrophages from aged mice were not as effective as adult macrophages in restoring the antibody response to the thymus-independent antigen dinitrophenyl-&adanylglycylglycyl (DHGG)-Ficoll. A possible reason for these differences could be that in the work of Heidrick and Makinodan (8) and ourselves T cells as well as macrophages are required whereas that of Nordin and Buchholz (9) is T cell independent, therefore different cellular interactions may be required in generating these responses. T cells obtained from aged mice are defective as accessory cells in the Fc fragment-induced polyclonal antibody response (200 PFC vs 75 PFC). That T cells from aged mice show reduced reactivity was indicated by their inability to substitute for adult T cells in the polyclonal antibody response. A reduced number of T cells in aged spleen does not appear to be responsible since the optimal response was obtained with the same B:T cell ratio as observed with adult T cells. Moreover, the low level of T-cell replacing factor (Fc)TRF production, in response to Fc fragments, is indicative of a T-cell lesion. Thoman et al. (30) have recently reported that the T-cell signal required in the Fc fragment-induced polyclonal antibody response could be replaced by a soluble factor (Fc)TRF, produced by T cells when incubated with Fc fragments. This soluble T-cell factor exerts its effects directly at the level of the responding B-cell population. Adult T cells were found to be approximately threefold more efficient in generating (Fc)TRF than aged T cells. The existence of a lesion in the T-cell compartment of aged mice in generating cell-mediated and humoral immune responseshas been extensively reported (3, 5, 11- 14, 34-36). This T-cell defect has been characterized as a reduction in T-cell

26

MORGAN, THOMAN,

AND WEIGLE

activity (34, 36) and/or an increase in suppressor T-cell function (3, 11, 12, 32). The role of aged T cells as suppressors in the system described here is unknown and must await further investigation. The inability of aged T cells to support the Fc fragment-induced polyclonal antibody response does not preclude the possibility that a defect exists in the B compartment as well. Aged splenic B cells were found to be defective in their ability to proliferate and produce polyclonal antibody in the presence of Fc fragments. Fc subfragments, derived from macrophage-processing of Fc fragments (19), which directly induce B cells to proliferate in the absence of accessory cells ( 19), produced only a minimal B-cell proliferation in aged spleen cell populations indicating a Bcell lesion. Moreover, adult T cells were unable to restore the T-cell-depleted aged B-cell polyclonal response to levels greater than those obtained with aged T cells. This finding suggests that although adult T cells are capable of providing a greater amount of help than aged T cells, the aged B cells are incapable of being activated to the level of adult B cells. In addition, aged T cells were found to be unable to suppress the Fc-mediated polyclonal antibody response of adult spleen cells. These results rule out the possibility of suppressor T cells being responsible for the low responses of aged mice. Other investigators have described lesions in the B-cell activity of aged mice as well (6, 13, 14, 35, 36). In addition, spleen cells from aged mice show reduced ability to respond to the adjuvant properties of Fc fragments (Morgan and Weigle, manuscript in preparation). Experimentation is currently in progress to define the cellular lesions for the inability of Fc to augment the humoral immune response of aged mice. In summary, we have shown that in an immune model where the cellular functions are known, lesions in both the B-cell and T-cell compartments exist in the aged C57BL/6 mouse. ACKNOWLEDGMENTS The authors wish to thank Nancy Kantor for excellent technical assistance and Janet Kuhns for secretarial expertise.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Waldorf, D. S. Wilkens, R. F., and Decker, J. L., JAMA 203, 111, 1968. Friedman, D., Kerser, V., and Globerson, A., Nature (London) 251, 545, 1974. Callard, R., and Basten, A., Cell. Immunol. 31, 13, 1977. DeLima, G., Wilkinson, M. J., Smith, G. S., and Walford, R. L., J. Gerontol. 29, 261, 1974. Segre, M., and Segre, D., J. Immunol. 116, 731, 1976. Callard, R. E., Basten, A., and Waters, L. K., Cell. Immunol. 31, 26, 1977. Nordin, A. A., and Adler, W. H., In “Developmental Immunobiology” (G. Siskind, S. Litwin and M. Weksler, Eds.), p. 215. Grune & Stratton, New York, 1979. 8. Heidrick, M. L., and Makinodan, T., J. Immunol. 111, 1502, 1973. 9. Nordin, A. A., and Buchholz, M. A., In “Techniques in Aging-Immunobiology Research” (W. H. Adler and A. A. Nordin, Eds.). CRC Press, New York, 1981, in press. 10. Callard, R. E., and Basten, A., Cell Immunol. 31, 13, 1977. 11. Goidl, E. A., Innes, J. B., and Weksler, M. E. J. Exp. Med. 144, 1037, 1976. 12. Weksler, M. E., Innes, J. B., and Goldstein, G., J. Exp. Med. 148, 996, 1978. 13. DeKruyff, R. H., Kim, Y. T., Siskind, G. W., and Weksler, M. E., J. Immunol. 125, 143, 1980. 14. Szewczuk, M. R., and Campbell, R. J., Nature (London) 286, 164, 1980. 15. Hallgren, H. M., Buckley, C. E., Gilbertsen, U.A., and Yunis, E. J., J. Immunof. 111, 1101, 1975. 16. Talal, N., In “Autoimmunity” (N. Talal, Ed.), p. 184. Academic Press, New York, 1977. 17. Berman, M. A., and Weigle, W. O., J. Enp. Med. 146, 241, 1977.

IMMUNE 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

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Morgan, E. L., and Weigle, W. O., J. Exp. Med. 150, 256, 1979. Morgan, E. L., and Weigle, W. 0.. J. Exp. Med. 151, 1, 1980. Morgan, E. L., and Weigle, W. O., J. Immunol. 124, 1330, 1980. Morgan, E. L., Walker, S. M., Thoman, M. L., and Weigle, W. O., J. Exp. Med. 152, 113, 1980. Morgan, E. L., Thoman, M. L., Walker, S. M., and Weigle, W. O., J. Immunol. 125, 1275, 1980. Morgan, E. L., and Weigle, W. O., J. Exp. Med., in press, 1981. Morgan, E. L., Thoman, M. L., and Weigle, W. O., J. Exp. Med. 153, 1161, 1981. Porter, R. R., Biochem. J. 73, 119, 1959. Spiegelberg, H. L., In “Textbook of Immunopathology” (P. A. Miescher and H. J. Mueller-Eberhard), P. 1101. Grune & Stratton, New York, 1976. Ly, I. A., and Mishell, R. I., J. Immunol. Methods 5, 239, 1974. Mueller, J., Brun del Re, G., Buerki, H., Keller, H. U., Hess, M. W., and Cottier, H., Eur. J. Immunol. 5, 270, 1975. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. Thoman, M. L., Morgan, E. L., and Weigle, W. O., J. Immunol., in press, 1981. Jerne, N. K., and Nordin, A. A., Science 140, 405, 1963. Kettman, J., and Dutton, R. W., J. Immunol. 104, 1558, 1970. Nordin, A. A., and Adler, W. H., In “Proceedings of the Fifth Irwin Strasburger Memorial Symposium on Immunology.” Grune & Stratton, New York, 1981, in press. Price, G. B., and Makinodan, T., J. Immunol. 108, 403, 1972. Friedman, D., Keiser, V., and Globerson, A., Nature (London) 251, 545, 1974. Doria, G., D’Agostaro, G., and Gararini, M., Cell Immunol. 53, 195, 1980.