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Bovine serum albumin humoral immune response in aged fischer 344 rats
Mechanisms of Ageing and Development, 16 (1981) 61-66 © Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
61
BOVINE SERUM ALBUMIN HUMORAL IMMUNE RESPONSE IN AGED FISCHER 344 RATS
PHYLLIS-JEAN KAPLAN, THOMAS J. CAPERNA and JUSTINE S. GARVEY* Biological Research Laboratories, Department of Biology, Syracuse University, Syracuse, N Y 13210 (U.S.A.)
(Received August 1, 1980)
SUMMARY The circulating antibody titers to bovine serum albumin (BSA) were determined for two groups of aged Fischer 344 rats obtained from the National Institute on Aging. One group received a single immunization of BSA in complete Freund's adjuvant, and the second group was immunized three times at two-week intervals with twice the antigen dosage. An age difference was observed during the peak response in both groups, the younger rats having higher titers; however, upon multiple injections the older rats show an enhanced response although significantly lower than the younger rats. All antibody titers increased at the same rate, yet measurable antibody always appeared first in younger animals.
INTRODUCTION Recently the National Institute on Aging established a colony of Fischer 344 (F344) rats. These rats are reared under strict, special environmental conditions so as to minimize external factors that are known to play a role in aging. Pathological findings correlated with age have been reported for this colony [1 ], and thus makes feasible the use of these rats for immunological studies. The intention of the present study is to investigate the kinetics of a humoral immune response to bovine serum albumin (BSA) after single and repeated injections. Since age-related changes in antibody production are found only in high responders to poly(GluS2LysaaTyr is) [2], it is noteworthy that Amerding et al. [3] have shown the F344 to respond well to BSA. Nordin and Adler [4] have suggested that aging should be considered as an.individual phenomenon since each animal ages at a different rate. In the following study, a complete profile of each rat's response was monitered, thereby allowing the "aging" process to be studied on an individual basis. *To whom reprint requests and correspondence should be addressed.
62 MATERIALS AND METHODS Animals
Two cohort groups of F344 rats were obtained from the National Institute on Aging colony (Charles River Breeding Laboratories, Wilmington, MA, U.S.A.). Each group consisted of pairs of male rats of the following ages Group A: 4, 8, 14, 20 and 26 months; group B: 2, 6, 12, 18, and 24 months. The 2- and 4-month-old rats were not from the aging colony, but were obtained from Charles River Breeding Laboratories at the same time as the colony rats. Pairs of rats of the san~e age were caged together in a separate animal room containing only colony rats and were maintained on a 12-h light cycle. Food and water were provided ad libitum. Immunization
Group A was hyperimmunized according to the method of Garvey et al. [6] with a 1 : 1 (v/v) emulsion of complete Freund's adjuvant (cFA, Difco) and 0.84% (w/v) BSA (Armour) in phosphate.buffered saline. The following amounts were injected: 0.05 ml in each hind foot pad (intradermaUy), 0.1 ml in each flank of hind leg (intramuscularly), 0.1 ml in each side of the lumbar region (subcutaneously) and 0.4 ml intraperitoneally. Group A rats received the above protocol for the primary injection. Two weeks later the same injection procedure without the foot pad route was followed. Between two and three weeks later the third set of injections, including foot pads, was administered. The injections were staggered between the age groups, but all bleedings after the third injection correspond to the same day. Group B rats were given a single primary injection as in the above protocol with 0.42% BSA in cFA. Collection o f sera
Experimental blood (1-1.5 ml) was collected by rupturing the retro-orbital plexus while the rats were etherized. Unimmunized F344 and Sprague-Dawley rats were bled periodically as above and by cardiac puncture for normal serum, which was then clarified by centrifugation at 40.000 g for 1 h at 4 °C. BSA iodination
The chloramine-T method as described by Garvey et al. [5] was used. After iodination, the reaction products were separated by Sephadex G-25 gel fdtration. Farr assay
A modification of the Farr assay [6] was performed to assess circulating antibody titers. For uniformity of all aspects of the assay, all samples of each group were analyzed at the same time. Briefly, assay tubes contained 0.4 ml of experimental serum in appropriate dilutions and 0.4 ml of 0.1 mg/m112SI-labelled bovine serum albumin ([12sI] BSA) in borate buffer. All experimental dilutions greater than 1/5 were made in borate buffer containing a 1/10 dilution of normal rat serum. The remaining experimental dilutions
63 were prepared in borate buffer alone. Normal serum controls consisted of the 1/10 dilution of normal rat serum and [l~sI] BSA. The BSA control contained only the 0.4 ml of [1251] BSA. All tubes were mixed, then incubated overnight at 0 - 4 °C. Saturated ammonium sulfate was added at a final con.centration of 50% to all but BSA control tubes. The samples were mixed, incubated for 30 min at 0 - 4 °C, and centrifuged for 1 h at 1200 g and 4 °C in a RC2-B Sorvall centrifuge. The supematants were decanted, and the precipitates were counted in a Nuclear Chicago gamma counter.
RESULTS Both groups A and B show an age-relatedness in circulating antibody titers after the initial immunization (Fig. 1). The younger rats always produce a higher titer. With the reduced dosage given group B, this phenomenon is more pronounced. The age difference throughout the peak response of the singly injected rats (group B) is significant (p <
I001~
r~ Z :3 0
(/1 nn B
-0
I
2
3
4
-LOG
DILUTION
5
6
Fig. 1. Antibody binding curves of a single bleeding determined by the Farr assay. At the time of bleeding, the only difference between the two groups was that group B received an antigen dose that was half that of group A.
64
5O
p
I
f
1
I
4 5
40 3.5 ~, 30 3
Group A J Rot NO. Age(month) --e--i 4 "-~- 3 8
2.5
-'*,'-4 I 5
"-o--7 --o-- 8 ~ 9 -~'- tO I
IO
Rot No/ 4gcD(motdh) ---*.--I 2 2 -'°--2
8 14
--~- 6
'_u 1.5
/
L
(15 - +0
0
I0
20
30
6 12 12 18 18 24 24
5 -'~- 6 7 --o-- 8 9 -~- ~
14 20 20 26 26
40
DAYS POST THIRD INJECTION
20
30
I
I
I
I
1
I
40
50
~
~
80
90
moo
DAYS POST INJECTION
Fig. 2. Composite 33% antigen-binding capacity (ABC-33) values for F344 rats with multiple immunizations (group A) and a single immunization (group B). Prior to the third injection in group A, not enough serum was obtained from rat 9 whereas rat 10 had not attained an ABC-33 value. In group B, no serum was available from rat 3 until day 20, and rat 9 was sacrificed on day 91 because of an obvious tumor. The statistical tests performed on the data were linear and multiple regression analyses.
0.01) as shown in Fig. 2. In the hyperimmunized group, however, titers show a reduced difference between old and young rats, but a significant difference between the various aged rats still exists (p < 0.01). Peak titers within each group were attained about the same day; and the kinetics of the rise to the peak response show no age-related distinctions, i.e. old and young produced antibody to BSA at the same rate.
DISCUSSION
The Farr assay is used to determine an antiserum's capability to bind antigen, thus assessing total antibody present as well as the avidity of the serum. The present study indicates that the younger rats have a higher antigen-binding capacity despite the different immunization protocols. Doria et al. [7] have shown that antiserum avidity is parabolically related to age with the highest avidity in the middle-aged mice. The findings in this report in conjunction with the findings of Doria et el. [7] suggest greater specific antibody concentrations are present in the young. Similarly, the older rats may exhibit their decreased antigen-binding capacity due to decreased antibody levels and/or decreased antiserum avidity. Despite the delayed appearance o f specific antibody and the lowered peak titers attained by the older rats, the rate of antibody production is the same in all ages. This
65 finding suggests that those cells producing antibody in the old are doing so at the same rate as the cells in the young, although there may be fewer responding cells in the older animal. Use of higher antigen doses and multiple injections cause the peak antibody titers of the older rats to be more similar to those of the younger rats. This is in accordance with the finding of Jaroslow et al. [8], that older dogs given repeated injections of T2 coliphage attained antibody levels comparable to those of the younger dogs. The authors attributed this to higher antigen levels aiding in the maintenance of the older dog's cell proliferation, either through increased progeny from each responding cell or increased numbers of responding cells. Despite the different experimental procedures employed, the group A findings are also in agreement with the reported results of Goullet and Kaufmann [9]. Using 3- and 22-month-old WAG rats, they have shown an age difference in antibody titers four to six weeks after several intramuscular immunizations with BSA in cFA. Although the disparity between the young and old during the peak response is reduced, a significant difference between the ages still exists. Price and Makinodan [10] reported that ten times as much antigen is needed to stimulate aged mice to their peak level of antibody production and that this level is still 100 times lower than the peak response of young mice. At lower antigen doses, as in group B, an age-relatedness appeared in F344 rat antibody production. The difference in titers was enhanced between young and old animals, with the older animals producing progressively lower amounts of antibody. These results are in agreement with other findings, i.e. a decline with age in immune responsiveness to exogenous antigen. Similar observations were found using various particulate and soluble antigens [11] and using different species; for example, humans [12], dogs [8], mice [10] and rats [2]. In all cases the antibody titer decreased with age. Perhaps, at still lower antigen concentrations, a latent period prior to antibody production or even a lack of antibody production might be observed. The decline in the humoral response may be attributed to a decrease in cellular ability to recognize or to be stimulated by the antigen. It has been shown that cellular cooperation diminishes with age; for example, T helper function decreases [13] and suppressor cell activity assumes a more active role [14]. Age-related changes in B cells have been noted by DeKruyff et al. [15]. Such findings may help to explain the age-related decline in antibody production with T-dependent antigens, such as BSA. Recently, Garvey et al. [16] demonstrated antigen retention in various tissues after intravenous injection of BSA in aged F344. Lower levels of antigen were retained in rats older than 12 months thereby reducing the amount of available antigen to mount an immune response. This finding along with the other established age phenomena, i.e. dysfunction in T regulatory roles as well as the recent idea of B cell malfunction, may aid in the explanation of the observed discrepancies between the young and old humoral immune response. Inasmuch as the antibody response is known to be govemed by the Q and R genes [17], these regions may also be involved in the age differences noted. In summary, age-related differences are observed during the peak response, the young have an earlier rise in titer than the old, and the rate of antibody production in all
66 ages is t h e same. These findings c o n f i r m p r e v i o u s h u m o r a l studies a n d are c o n s i s t e n t w i t h c u r r e n t t h e o r i e s o n aging.
ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y G r a n t No. A G 0 1 1 1 f r o m t h e N a t i o n a l I n s t i t u t e s o f Health.
REFERENCES 1 G. L. Coleman, S. W. Barthold, G. W. Osbaldiston, S. J. Foster and A. M. Jonas, Pathological changes during aging in barrier-reared Fischer 344 male rats. Jr. GerontoL, 32 (1977) 258-278. 2 H. W. Kunz, T. J. Gill, III and J. M. Corson, Effects of age and genetic background on the antibody response in inbred rats. Gerontologia, 20 (1974) 8 8 - 1 0 1 . 3 D. Amerding, D. H. Katz and B. Benacerraf, Immune response genes in inbred rats. I. Analysis of responder status to synthetic polypeptides and low doses of bovine serum albumin. Immunogenetics, 1 (1974) 329-339. 4 A. L. Nordin and W. H. Adler, The effect of aging on in vitro cellular interactions. In G. W. Siskind, S. D. Litwin and M. E. Weksler (eds.), Developmental lmmunobiology (Strasberger Memorial Seminar), Grune and Stratton, New York, 1979, p. 215. 5 J. S. Garvey, N. E. Cremer and D. H. Sussdorf, Methods in Immunology, 3rd edn., W. A. Benjamin, Inc., Reading, MA, 1977. 6 P. Minden and R. S. Farr, Ammonium sulfate method to measure antigen binding capacity. In D. M. Weir (ed.), Handbook of Experimental Immunology, Blackwell Scientific, Oxford, 1978, pp. 13.1-13.22. 7 G. Doria, G. D'Agostoro and A. Poretti, Age dependent variations of antibody activity. Immunology, 35 (1978) 6 0 1 - 6 1 1 . 8 B. N. Jaroslow, K. M. Suhrbier and T. E. Fritz, Decline and restoration of antibody-forming capacity in aging beagle dogs. J. Immunol., 112 (1974) 1467-1476. 9 P. Goullet et H. Kaufman, Etudes sur la rdponse immunitaire selon l'flge chez le rat. Gerontologia, 10 (1964/65) 7 6 - 8 5 . 10 G. B. Price and T. Makinodan, Immunological deficiencies in senescence. I. Characterization of intrinsic deficiencies. Z Immunol., 108 (1972) 4 0 3 - 4 1 2 . 11 R. E. Callard and A. Basten, Immune function in aged mice. IV. Loss of T cell and B cell function in thymus-dependent antibody responses. Eur. J. ImmunoL, 8 (1978) 5 5 2 - 5 5 8 . 12 M. Rowley, H. Buchanan and I. R. Mackay, Reciprocal change with age in antibody to extrinsic and intrinsic antigens. Lancet, ff (1968) 2 4 - 2 6 . 13 M. M. B. Kay, Effect of age on T cell differentiation. Fed. Proc., 37 (I 978) 1241-1244. 14 S. K. Singhal, J. C. Roder and A. K. Duwe, Suppressor dells in immunosenescence. Fed. Proc., 37 (1978) 1245-1252. 15 R. H. DeKruyff, Y. T. Kim, G. W. Siskind and M. E. Weksler, Age related changes in the in vitro immune response: increased suppressor activity in immature and aged mice. Z ImmunoL, 125 (1980) 142-147. 16 J. S. Garvey, J. W. Shafer and T. J. Caperna, Antigen handling in relation to ageing. Mech. Ageing Dev., 12 (1980) 2 8 7 - 3 0 3 . 17 T. J. Gill III, H. W. Kunz, D. J. Stechschulte and K. F. Austen, Genetic and cellular factors in the immune response. I. Genetic control of the antibody response to poly(GluS2LysaaTyr Is) in the inbred rat strains ACI and F344. J. Immunol., 105 (1970) 14-28.
61
BOVINE SERUM ALBUMIN HUMORAL IMMUNE RESPONSE IN AGED FISCHER 344 RATS
PHYLLIS-JEAN KAPLAN, THOMAS J. CAPERNA and JUSTINE S. GARVEY* Biological Research Laboratories, Department of Biology, Syracuse University, Syracuse, N Y 13210 (U.S.A.)
(Received August 1, 1980)
SUMMARY The circulating antibody titers to bovine serum albumin (BSA) were determined for two groups of aged Fischer 344 rats obtained from the National Institute on Aging. One group received a single immunization of BSA in complete Freund's adjuvant, and the second group was immunized three times at two-week intervals with twice the antigen dosage. An age difference was observed during the peak response in both groups, the younger rats having higher titers; however, upon multiple injections the older rats show an enhanced response although significantly lower than the younger rats. All antibody titers increased at the same rate, yet measurable antibody always appeared first in younger animals.
INTRODUCTION Recently the National Institute on Aging established a colony of Fischer 344 (F344) rats. These rats are reared under strict, special environmental conditions so as to minimize external factors that are known to play a role in aging. Pathological findings correlated with age have been reported for this colony [1 ], and thus makes feasible the use of these rats for immunological studies. The intention of the present study is to investigate the kinetics of a humoral immune response to bovine serum albumin (BSA) after single and repeated injections. Since age-related changes in antibody production are found only in high responders to poly(GluS2LysaaTyr is) [2], it is noteworthy that Amerding et al. [3] have shown the F344 to respond well to BSA. Nordin and Adler [4] have suggested that aging should be considered as an.individual phenomenon since each animal ages at a different rate. In the following study, a complete profile of each rat's response was monitered, thereby allowing the "aging" process to be studied on an individual basis. *To whom reprint requests and correspondence should be addressed.
62 MATERIALS AND METHODS Animals
Two cohort groups of F344 rats were obtained from the National Institute on Aging colony (Charles River Breeding Laboratories, Wilmington, MA, U.S.A.). Each group consisted of pairs of male rats of the following ages Group A: 4, 8, 14, 20 and 26 months; group B: 2, 6, 12, 18, and 24 months. The 2- and 4-month-old rats were not from the aging colony, but were obtained from Charles River Breeding Laboratories at the same time as the colony rats. Pairs of rats of the san~e age were caged together in a separate animal room containing only colony rats and were maintained on a 12-h light cycle. Food and water were provided ad libitum. Immunization
Group A was hyperimmunized according to the method of Garvey et al. [6] with a 1 : 1 (v/v) emulsion of complete Freund's adjuvant (cFA, Difco) and 0.84% (w/v) BSA (Armour) in phosphate.buffered saline. The following amounts were injected: 0.05 ml in each hind foot pad (intradermaUy), 0.1 ml in each flank of hind leg (intramuscularly), 0.1 ml in each side of the lumbar region (subcutaneously) and 0.4 ml intraperitoneally. Group A rats received the above protocol for the primary injection. Two weeks later the same injection procedure without the foot pad route was followed. Between two and three weeks later the third set of injections, including foot pads, was administered. The injections were staggered between the age groups, but all bleedings after the third injection correspond to the same day. Group B rats were given a single primary injection as in the above protocol with 0.42% BSA in cFA. Collection o f sera
Experimental blood (1-1.5 ml) was collected by rupturing the retro-orbital plexus while the rats were etherized. Unimmunized F344 and Sprague-Dawley rats were bled periodically as above and by cardiac puncture for normal serum, which was then clarified by centrifugation at 40.000 g for 1 h at 4 °C. BSA iodination
The chloramine-T method as described by Garvey et al. [5] was used. After iodination, the reaction products were separated by Sephadex G-25 gel fdtration. Farr assay
A modification of the Farr assay [6] was performed to assess circulating antibody titers. For uniformity of all aspects of the assay, all samples of each group were analyzed at the same time. Briefly, assay tubes contained 0.4 ml of experimental serum in appropriate dilutions and 0.4 ml of 0.1 mg/m112SI-labelled bovine serum albumin ([12sI] BSA) in borate buffer. All experimental dilutions greater than 1/5 were made in borate buffer containing a 1/10 dilution of normal rat serum. The remaining experimental dilutions
63 were prepared in borate buffer alone. Normal serum controls consisted of the 1/10 dilution of normal rat serum and [l~sI] BSA. The BSA control contained only the 0.4 ml of [1251] BSA. All tubes were mixed, then incubated overnight at 0 - 4 °C. Saturated ammonium sulfate was added at a final con.centration of 50% to all but BSA control tubes. The samples were mixed, incubated for 30 min at 0 - 4 °C, and centrifuged for 1 h at 1200 g and 4 °C in a RC2-B Sorvall centrifuge. The supematants were decanted, and the precipitates were counted in a Nuclear Chicago gamma counter.
RESULTS Both groups A and B show an age-relatedness in circulating antibody titers after the initial immunization (Fig. 1). The younger rats always produce a higher titer. With the reduced dosage given group B, this phenomenon is more pronounced. The age difference throughout the peak response of the singly injected rats (group B) is significant (p <
I001~
r~ Z :3 0
(/1 nn B
-0
I
2
3
4
-LOG
DILUTION
5
6
Fig. 1. Antibody binding curves of a single bleeding determined by the Farr assay. At the time of bleeding, the only difference between the two groups was that group B received an antigen dose that was half that of group A.
64
5O
p
I
f
1
I
4 5
40 3.5 ~, 30 3
Group A J Rot NO. Age(month) --e--i 4 "-~- 3 8
2.5
-'*,'-4 I 5
"-o--7 --o-- 8 ~ 9 -~'- tO I
IO
Rot No/ 4gcD(motdh) ---*.--I 2 2 -'°--2
8 14
--~- 6
'_u 1.5
/
L
(15 - +0
0
I0
20
30
6 12 12 18 18 24 24
5 -'~- 6 7 --o-- 8 9 -~- ~
14 20 20 26 26
40
DAYS POST THIRD INJECTION
20
30
I
I
I
I
1
I
40
50
~
~
80
90
moo
DAYS POST INJECTION
Fig. 2. Composite 33% antigen-binding capacity (ABC-33) values for F344 rats with multiple immunizations (group A) and a single immunization (group B). Prior to the third injection in group A, not enough serum was obtained from rat 9 whereas rat 10 had not attained an ABC-33 value. In group B, no serum was available from rat 3 until day 20, and rat 9 was sacrificed on day 91 because of an obvious tumor. The statistical tests performed on the data were linear and multiple regression analyses.
0.01) as shown in Fig. 2. In the hyperimmunized group, however, titers show a reduced difference between old and young rats, but a significant difference between the various aged rats still exists (p < 0.01). Peak titers within each group were attained about the same day; and the kinetics of the rise to the peak response show no age-related distinctions, i.e. old and young produced antibody to BSA at the same rate.
DISCUSSION
The Farr assay is used to determine an antiserum's capability to bind antigen, thus assessing total antibody present as well as the avidity of the serum. The present study indicates that the younger rats have a higher antigen-binding capacity despite the different immunization protocols. Doria et al. [7] have shown that antiserum avidity is parabolically related to age with the highest avidity in the middle-aged mice. The findings in this report in conjunction with the findings of Doria et el. [7] suggest greater specific antibody concentrations are present in the young. Similarly, the older rats may exhibit their decreased antigen-binding capacity due to decreased antibody levels and/or decreased antiserum avidity. Despite the delayed appearance o f specific antibody and the lowered peak titers attained by the older rats, the rate of antibody production is the same in all ages. This
65 finding suggests that those cells producing antibody in the old are doing so at the same rate as the cells in the young, although there may be fewer responding cells in the older animal. Use of higher antigen doses and multiple injections cause the peak antibody titers of the older rats to be more similar to those of the younger rats. This is in accordance with the finding of Jaroslow et al. [8], that older dogs given repeated injections of T2 coliphage attained antibody levels comparable to those of the younger dogs. The authors attributed this to higher antigen levels aiding in the maintenance of the older dog's cell proliferation, either through increased progeny from each responding cell or increased numbers of responding cells. Despite the different experimental procedures employed, the group A findings are also in agreement with the reported results of Goullet and Kaufmann [9]. Using 3- and 22-month-old WAG rats, they have shown an age difference in antibody titers four to six weeks after several intramuscular immunizations with BSA in cFA. Although the disparity between the young and old during the peak response is reduced, a significant difference between the ages still exists. Price and Makinodan [10] reported that ten times as much antigen is needed to stimulate aged mice to their peak level of antibody production and that this level is still 100 times lower than the peak response of young mice. At lower antigen doses, as in group B, an age-relatedness appeared in F344 rat antibody production. The difference in titers was enhanced between young and old animals, with the older animals producing progressively lower amounts of antibody. These results are in agreement with other findings, i.e. a decline with age in immune responsiveness to exogenous antigen. Similar observations were found using various particulate and soluble antigens [11] and using different species; for example, humans [12], dogs [8], mice [10] and rats [2]. In all cases the antibody titer decreased with age. Perhaps, at still lower antigen concentrations, a latent period prior to antibody production or even a lack of antibody production might be observed. The decline in the humoral response may be attributed to a decrease in cellular ability to recognize or to be stimulated by the antigen. It has been shown that cellular cooperation diminishes with age; for example, T helper function decreases [13] and suppressor cell activity assumes a more active role [14]. Age-related changes in B cells have been noted by DeKruyff et al. [15]. Such findings may help to explain the age-related decline in antibody production with T-dependent antigens, such as BSA. Recently, Garvey et al. [16] demonstrated antigen retention in various tissues after intravenous injection of BSA in aged F344. Lower levels of antigen were retained in rats older than 12 months thereby reducing the amount of available antigen to mount an immune response. This finding along with the other established age phenomena, i.e. dysfunction in T regulatory roles as well as the recent idea of B cell malfunction, may aid in the explanation of the observed discrepancies between the young and old humoral immune response. Inasmuch as the antibody response is known to be govemed by the Q and R genes [17], these regions may also be involved in the age differences noted. In summary, age-related differences are observed during the peak response, the young have an earlier rise in titer than the old, and the rate of antibody production in all
66 ages is t h e same. These findings c o n f i r m p r e v i o u s h u m o r a l studies a n d are c o n s i s t e n t w i t h c u r r e n t t h e o r i e s o n aging.
ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d b y G r a n t No. A G 0 1 1 1 f r o m t h e N a t i o n a l I n s t i t u t e s o f Health.
REFERENCES 1 G. L. Coleman, S. W. Barthold, G. W. Osbaldiston, S. J. Foster and A. M. Jonas, Pathological changes during aging in barrier-reared Fischer 344 male rats. Jr. GerontoL, 32 (1977) 258-278. 2 H. W. Kunz, T. J. Gill, III and J. M. Corson, Effects of age and genetic background on the antibody response in inbred rats. Gerontologia, 20 (1974) 8 8 - 1 0 1 . 3 D. Amerding, D. H. Katz and B. Benacerraf, Immune response genes in inbred rats. I. Analysis of responder status to synthetic polypeptides and low doses of bovine serum albumin. Immunogenetics, 1 (1974) 329-339. 4 A. L. Nordin and W. H. Adler, The effect of aging on in vitro cellular interactions. In G. W. Siskind, S. D. Litwin and M. E. Weksler (eds.), Developmental lmmunobiology (Strasberger Memorial Seminar), Grune and Stratton, New York, 1979, p. 215. 5 J. S. Garvey, N. E. Cremer and D. H. Sussdorf, Methods in Immunology, 3rd edn., W. A. Benjamin, Inc., Reading, MA, 1977. 6 P. Minden and R. S. Farr, Ammonium sulfate method to measure antigen binding capacity. In D. M. Weir (ed.), Handbook of Experimental Immunology, Blackwell Scientific, Oxford, 1978, pp. 13.1-13.22. 7 G. Doria, G. D'Agostoro and A. Poretti, Age dependent variations of antibody activity. Immunology, 35 (1978) 6 0 1 - 6 1 1 . 8 B. N. Jaroslow, K. M. Suhrbier and T. E. Fritz, Decline and restoration of antibody-forming capacity in aging beagle dogs. J. Immunol., 112 (1974) 1467-1476. 9 P. Goullet et H. Kaufman, Etudes sur la rdponse immunitaire selon l'flge chez le rat. Gerontologia, 10 (1964/65) 7 6 - 8 5 . 10 G. B. Price and T. Makinodan, Immunological deficiencies in senescence. I. Characterization of intrinsic deficiencies. Z Immunol., 108 (1972) 4 0 3 - 4 1 2 . 11 R. E. Callard and A. Basten, Immune function in aged mice. IV. Loss of T cell and B cell function in thymus-dependent antibody responses. Eur. J. ImmunoL, 8 (1978) 5 5 2 - 5 5 8 . 12 M. Rowley, H. Buchanan and I. R. Mackay, Reciprocal change with age in antibody to extrinsic and intrinsic antigens. Lancet, ff (1968) 2 4 - 2 6 . 13 M. M. B. Kay, Effect of age on T cell differentiation. Fed. Proc., 37 (I 978) 1241-1244. 14 S. K. Singhal, J. C. Roder and A. K. Duwe, Suppressor dells in immunosenescence. Fed. Proc., 37 (1978) 1245-1252. 15 R. H. DeKruyff, Y. T. Kim, G. W. Siskind and M. E. Weksler, Age related changes in the in vitro immune response: increased suppressor activity in immature and aged mice. Z ImmunoL, 125 (1980) 142-147. 16 J. S. Garvey, J. W. Shafer and T. J. Caperna, Antigen handling in relation to ageing. Mech. Ageing Dev., 12 (1980) 2 8 7 - 3 0 3 . 17 T. J. Gill III, H. W. Kunz, D. J. Stechschulte and K. F. Austen, Genetic and cellular factors in the immune response. I. Genetic control of the antibody response to poly(GluS2LysaaTyr Is) in the inbred rat strains ACI and F344. J. Immunol., 105 (1970) 14-28.