Immune function in aged mice

CELLULAR

IMMUNOLOGY

31, 26-36 (1977)

Immune

Function II. B-Cell

E. CALLARD,

ROBIN

Iw~zz~~zology

ANTONY

in Aged Mice Function

BASTEN,

AND

LLEWYN

Unit, Department of Bzctcriology, Univwsity Sydsaey, New Scuth Wales, 2006, Australia Received

K.

WATERS

of Sydney,

Novcwzzbcr 5, 1976

A loss of B-cell function in old mice was demonstrated by measuring the in vitro response of lymphoid cells to the B-cell polyclonal activator, LPS (lipopolysaccharide) , and the in viva response to the thymus-independent antigen, pneumococcal polysaccharide type III (SIII). The reduced mitogenic reactivity of lymphoid cells from old compared with young mice could not be explained by a shift in kinetics of the responding cells. When LPS cultures were carried out in the presence of colchicine, fewer cells from old mice were found to respond to the mitogenic signal. Th’e total number of B cells assessed by label!ing with Neither anti-immunoglobulin serum or antigenantibody complexes was not decreased in old animals. Taken together, these results are consistent with a qualitative rather than a quantitative loss of B-cell function with age. They did not, however, exclude the possibility of depletion of an LPS-reactive subpopulation of B cells. Since the number of LPS-reactive cells could not be determined directly, the antibody response of old mice to SIII was investigated. The decreased level of antibody production by old mice to SIII was not due to a shift in kinetics of the responding cells. Extracellular influences were excluded by showing that the reduced responsiveness of old spleen cells persisted after adoptive transfer into young irradiated recipients. Furthermore, pretreatment of cells from old mice with anti-Thy.1 serum and ccmplement before transfer did not enhance their antibody-forming potential. The loss of B-cell activity with age cou!d not, therefore, be explained in terms of an increase in T-cell-dependent suppressive effects. Support for an intrinsic defect in the B cell itself came from the demonstration of similar numbers of SIII-binding cells in normal spleens from old and young mice. Following immunisation, a shift toward low intensity binding cells was observed in spleens from both old and young mice. This shift was, however, less pronounced in the case of old cells, which is consistent with an age-related decline in transformation potential of antibody-forming-cell precursors. The conc!usion was, therefore, reached that the reduction with age in B-cell as well as T-cell function is due to a qualitative rather than a quantitative defect in lymphocytes th’emselves.

INTRODUCTION The data presented in the previous paper ( 1) indicated that the decline in cellmediated immunity associated with age is probably due to a qualitative defect in the T cell itself. The aim of this paper is to utilize similar approaches to analyse the mechanism of reduced antibody-forming potential known to accompany old age (2, 3). In theory, this could be due to an abnormality not only in B cells per se but, in addition, in T cells and/or macrophages which are known to play a vital role in many humoral immune responses (4, 5). B-cell function was, therefore, 26 Copyright All rights

@ 1977

by

Academic

of reproduction in any

Press, Inc. form reserved.

ISSN

0008

8749

B-CELL

FUNCTIOX

IX

OLD

MICE

27

assessed by measuring the response of spleen cells from old mice to the T-cellindependent antigen (6)) pneumococcal polysaccharide (SIII) ’ and the polyclonal mitogen, lipopolysaccharide (LPS) . The reduced responses observed could not be explained in terms of cell depletion since the total number of B cells and the number of specific SIII-binding cells were normal. The conclusion was reached that the age-related decline in humoral as well as cell-mediated immunity is explained better by a qualitative rather than a quantitative defect in lymphocyte function. MATERIALS

AND

METHODS

A~zinzals. Male and female CBA/H mice were used. Young and old mice were tested at 3-6 and 22-24 months of age, respectively. Their origin and rnainteilance were described in the preceding paper (1). Ccl1 s~pcnsions. Cells from spleens and mesenteric lymph nodes were teased through an SO-mesh stainless-steel sieve into cold medium 199 containing 5% foetal calf serum (FCS) (Commonwealth Serum Laboratories, Melbourne, Australia). Bone nlarrow cells were expressed from femurs and tibiae by nleans of a syringe and a 25-gauge needle containing medium 199 supplemented with 5% FCS. The cells were then dispersed by a single passage through a 21-gauge needle and washed once before counting. Viability deternlined by trypan blue exclusion was consistently between 80 and 90%. Removal of dead cells, cell debris, and erythrocytes. For cell labelling experiments, lymphoid cells free of debris and erythrocytes and with a viability in excess of 98% were obtained essentially as described by von Boehmer and Shortnlan (7). Mitogens and LPS clIItI[Yes. The source of lipolysaccharide iron1 Esclzerichia coli (LPS) and the technique of LPS cultures were described in the preceding paper ( 1) . Antigens. Pneuniococcal polysaccharide type III (SIII) was prepared by the nlethod of Heidelberger ef al. (8). For immunisation, it was dissolved in 0.14 hJ NaCl and injected intraperitoneally. For iodination, the p-hydroxybenzyl derivative (9) was used. Fowl y-globulin (FYG) was obtained by the method of Miller and Warner (10) and was absorbed with niouse thymus cells to remove nonspecific antilymphocyte activity. P?teparation of iodinated reagents. SIII and FYG were labelled with lz51 (Radiochemical Centre, Amersham, England, Catalog No. IMS 30) by the chloramine-T niethod of Greenwood et al. (11). Specific activity was 10-15 pCi/pg for each antigen. Preparation of antisera. Anti-immunoglobulin and anti-Thy-l.2 sera were prepared as described in the previous paper (1). Anti-FUG sertum was produced according to the method of Basten et al. ( 12). Irradiation. Mice were exposed to total body irradiation from a Cobalt-60 source. The dose used was 800 rad at a dose rate of 40 rad/min. In order to 1 Abbreviations used : ABC, antigen-binding cells ; AFCP, antibody-forming-cell precursors ; FcR, receptors for the Fc portion of immunoglobulin; FCS, foetal calf serum; FYG, fowl r-globulin ; FITC-sheep anti-Rig, fluorescein isothiocyanate-conjugated sheep anti-rabbit immunoglobulin; LPS, lipopolysaccharide from Eschcrickiu coli; PBS, phosphate-buffered saline, pH 7.2; PFC, plaque-forming cells; rabbit-F (ab) ‘2 anti-MIg, F (ab) ‘a fraction of rabbit anti-mouse immunoglobulin; SRBC, sheep red blood cells; SIII, pneumococcal polysaccharide type III ; [‘“I] FYG, l”I-labelled fo\vl y-globulin.

28

CALLARD,

BASTEN

AND

WATERS

ensure uniformity of tissue distribution of irradiation, the animals were rotated in a Perspex box at 1.5 rpm around a vertical axis in the horizontal beam from the source. The box was enclosed in a ventilated lead case under conditions of maximum backscatter. The beam was filtered with shaped lead disks to a close uniformity across the beam of greater than 96%. Radioautography. Radioautographs were prepared as described in the previous paper (1). Qua&it&ion of B cells. The total number of B cells was estimated by labelling of immunoglobulin or Fc receptors on the cell membrane. Immunoglobulin-bearing cells were detected by an immunofluorescent sandwich technique. Two million cells were incubated sequentially with 20 ~1, respectively, of rabbit-F (ab) ‘2 anti-MIg (1) and FITC-sheep anti-Rig (Burroughs Wellcome, Beckenham, England). Each step was carried out for 30 min on ice in a total volume of 100 ~1 of medium 199 (Commonwealth Serum Laboratories, Melbourne, Australia) containing 20% FCS and 0.17 o sodium azide. After final incubation, the cells were washed twice, mounted on a glass slide in 90% glycerol in phosphate-buffered saline (PBS) pH 7.2, and examined with a Zeiss ultraviolet microscope using B12/4 exciter filter. The percentage of fluorescent-labelled small mononuclear (B) cells was determined by counting 500-1000 cells per slide. Fc receptor (FcR)-bearing B lymphocytes were detected by binding of antibody-antigen complexes as described by Basten et al. (12). Briefly, 5 x 10G cells were incubaetd with 40 ~1 of a high titre CBA anti-FYG serum for 30 min at 37°C in a total volume of 0.4 ml of medium 199 containing 5% FCS. These were then spun down and resuspended in 0.2 ml of medium containing 0.5 pg of l”“I]FYG. After a further incubation at 37°C for 30 min unbound label was removed by centrifugation through FCS and the cells were smeared on gelatincoated slides for radioautography. The percentage of Fc receptor-bearing cells was obtained by counting 500-1000 cells per slide. Under these conditions, the great majority of FcR-positive cells are known to be B lymphocytes (12). cells were detected Detection of antigen-binding cells. SIII-antigen-binding as described by Byrt and Ada (13). Briefly, 5 x loo spleen cells were incubated with 0.5 pg of 1”51-labelled SIII for 30 min on ice in a total volume of 0.4 ml of medium 199 containing 5% FCS and O.l/OO sodium azide. Unbound label was removed by centrifugation through discontinuous gradients of FCS and the cell pellet was smeared on gelatin-coated slides for radioautography. The percentage of SIII-binding cells was estimated by counting a total of 5 X 10” cells/slide. plaque-forming cells (PFC) were Detection of plaque-forming cells. SIII detected by the method of Cunningham and Szenberg (14). Sheep red blood cells (SRBC) were sensitized by incubating one volume of 50% SRBC with two volumes of SIII supernatant for 1 hr at 37°C (15). Sensitized cells were washed three times in 0.02 M PBS before use. Fresh guinea pig serum absorbed with SIII-coated SRBC was used as a source of complement. Treatment zuitjz anti-thy-l.2 serus?z.T-cell-depleted spleen cell preparations were obtained by treatment with anti-Thy-l.2 serum and complement. One milliliter of anti-Thy-l.2 serum prepared as described previously (1) was added to lo8 spleen cells at a concentration of 2.5 X loo cells/ml. After an incubation period of 30 min at 37”C, the cells were washed twice and exposed at a concentration of 5 X 10’ cells/ml to agarose-absorbed complement diluted 1 : 6 for a further 30 min at 37°C.

B-CELL

FUKCTJON

IN

OLD

29

XTCE

Sfatistics. Analyses of PFC responses were performetl on log-transformed data as this treatment has been shown to norndise the distribution of PFCs (16). l’ercentage data were arc sine transformed before analysis. Student’s t-test and analyses of variance were carried out as descibctl by Snetlecor and Cochran ( 17). RESULTS A.

h’inctics

of the LPS

1Zespon.w of Old Compared

z&a

Young

Spleen

Cells

Dose-response curves for spleen cells from young and old mice were obtained by using different concentrations of LPS (Fig. 1A). Old spleen cells were less responsive than young at each dose of LPS (P < 0.001). A maximal response was obtained for both young and old cells with 2.5 pg/ml of LPS which was, therefore, used routinely in all future experiments. Time-response curves were obtained by harvesting cultures at different time intervals. Significant incorporation of [“HI thytnidine above background was observed with young cells after 24 hr in culture with LPS (Fig. 1B). Cells from old mice, on the other hand, did not incorporate significant amounts of isotope until 36 hr after the beginning of culture indicating a lag period of 12 hr in comparison with young cells. A peak response was obtained at 60 hr for both young and old cells, but old cells incorporated less [“HI thymidine at each time of assay up to 72 hr. After this, the response of both young and old cells declined rapidly and no difference between them was evident. B.

LPS

Stimulation

in tlze Presence

of Colchirine

Colchicine prevents cell division by inhibiting spindle formation but permits the DNA synthesis necessary for the first mitotic cycle (1s). Incorporation of [“H ] thymidine by lymphocytes activated with LPS in the presence of colchicine is, therefore, proportional to the number of cells activated by the mitogenic signal. Spleen cells from young and old mice were cultured with 2.5 pg/ml of LPS in the presence of 10m5M colchicine. Isotope was added at the beginning of the culture period and the cells were harvested after 24, 4S, and 72 hr (Fig. 2). Incorporation of [“H] thymitline reached a n~aximum after 4S hr in cultures of both young rA

6.0

6.0

E D 0 5.0 4

4.0

10 MICROGRAMS

100 OF

LPS

200 PER

CULTURE

24 HOURS

48 IN

72

96

CULTURE

FIG. 1. LPS dose-response (,4) and time-response (B) curves of spleen cells from young (0) and old ( 0 ) mice. Each point represents the mean response of ceils from six mice *95% confidence limits.

30

CALLARD,

BASTEN

AND

WATERS

5.0 -

3.0

48

24 HOURS

IN

72

CULTURE

FIG. 2. LPS response of spleen cells frcm young (0) and old ( l ) mice cultured in the presence of 10e5 M colchicine. Each point represents the mean of six cultures “95% confidence limits.

and old cells and then declined slightly at 72 hr. The decline suggested release of incorporated isotope from some of the cells stimulated to divide by LPS and may have been due to cell death. Old cells incorporated less isotope at each time of culture indicating the presence of fewer cells in the spleens of old mice capable of responding to LPS by initiating cell division. In order to determine whether this was due to depletion of reactive B cells in the spleens of old mice, the total number of B cells was compared with that from young mice. C.

Quantitation

of B Cells

Individual spleens, mesenteric lymph nodes, and bone marrow suspensions were obtained from young and old mice, .and the total number of small mononuclear cells was counted in each tissue. The proportion of immunoglobulin-bearing small lymphocytes was assessed by a fluorescent sandwich technique. The F(ab)‘z fragment of rabbit anti-MIg was used to ensure that binding did not occur to Fc TABLE Quantitation Source of cells

Spleen Lymph node Bone marrow

of B Cells in Lymphoid Donor mice

Total number of lymphocytes/ organ (X 10-9

Old Young Old Young Old Young

170 f 130 f -b -

4a 3

1 Tissue from Young and Old Mice Ig-bearing cells (%I

FcR-bearing cells (%)

55 f 3 42 + 1 21 +3 1.5 f 1 18 f 2 5fl

52 f 1 40 z!z 2 -

Number of B cells/spleen (x10-6)

89 f 53 f -

5 1

a Mean value from 12 mice I!Z SE. b Not done; mesenteric nodes in old mice were ill defined and so embedded in fat that complete excision could not be ensured and no meaningful comparison of total cell numbers could be made between young and old mice.

B-CELL

FUi’iCTION

IN

OLD

31

MICE

receptors on the cell membrane. As shown in Table 1, an increase rather than a decrease was observed in both the proportion and absolute numbers of B cells as measured by this technique. The greater proportion of B cells in old mice was shown to be statistically significant (P < 0.001) by a two-way analysis of variance and occurred to a similar degree in spleen and lymph node. Fc receptors appear before membrane-associated Ig determinants during B-cell ontogeny (19). Since extrameclullary haemopoiesis in old spleens could have resulted in an increased ratio of FcK-bearing/Ig-bearing cells, it was important to check for any discrepancy between the proportion of cells displaying these two determinants in lymphoid tissue from young and old mice. As shown in Table 1, none was observed. Taken together, these results fail to support the notion of cell depletion as an explanation for decreased B-cell responsiveness with age. They do not, however, exclude the possibility of depletion of a subpopulation of reactive B cells. To resolve this, the response of young and old mice to the thymus-independent antigen SIII was assessed in relation to the number of available antibodyforming cell precursors. D.

Antibody

Response of Old Mice to the T123,‘1i~2(s-IndependentAntigen,

SIII

Intact young and old mice were challenged with different amounts of SIII and the numbers of direct PFC were determined 5 days later (Fig. 3A). The antibody response of old mice to immunogenic closes of SIII (l-10 pg) was significantly less than that of youilg mice. A maximum response was elicited with 5 pg of SIII in both young and old mice. No indirect PFC were detected in any animal. Doses in excess of 10 pg were tolerogenic. The difference between old and young animals might have been due to a shift in kinetics of antibody production. Direct PFC were, therefore, determined at various times after immunisation of young and old mice with an optimal (5 pg) dose of SIII. As shown in Fig. 3B, the response of old mice was significantly less than that of young mice (P < 0.01) at each time of assay. Furthermore, the peak response occurred in both groups between Days 5 and 6.

2

4-

K 03 0" -I 2

15 2 DOSE

4 6 6 10 125 OF WI IN MICROGRAMS

1 2 DAYS

I 5 AFTER

I 10 lMMlJNlSATlON

I 15

FIG. 3. Dose-response (A) and time-response (B) curves of the direct PFC response young (0) and old ( l ) mice to SIII. Each point represents the mean response five to seven mice 295% confidence limits.

of of

32

CALLARD,

BASTEN

AND

TABLE

WATERS

2

Comparison of Anti-S111 Antibody Responses of Young and Old Spleeu Cells following Adoptive Transfer into Young Irradiated Recipients Source of donor spleen cells

Number of irradiated recipients

Old mice Young mice

8 8

a Arithmetic

E.

mean f

standard

Direct anti-S111 PFC/spleen at 5 days 1,500 f 18,300 f

4ooa 7,000

error.

Antibody Response of Old Spleen Cells to SIII Irradiated Recipients

after Transfer

into Young

The reduced response of old mice to SIII could have been due either to an abnormality in microenvironments within the lymphoid tissue or to a primary defect in B cells themselves. To distinguish between the two, an adoptive transfer system was used. Forty million spleen cells from young or old mice were injected intravenously into young (3-6 months old) irradiated recipients which were then challenged with 5 pg of SIII. The number of PFC per spleen was quantitated 5 days later (Table 2). The antibody response of spleen cells from old mice was significantly less than that of spleen cells obtained from young mice (P < 0.001). Furthermore, the difference was comparable to that obtained in intact animals. The reduced antibody-forming potential of old mice, therefore, appeared to be due to an abnormality in the lymphoid cells rather than to extracellular host influences. F.

Antibody Response of Old Spleen Cells to SIII Thy.1 Serum

after Treatment

with Anti-

Baker et al. (20) have demonstrated that T cells can exert a regulatory influence on the adoptive immune response to SIII. Thus, the reduction in antibody production to SIII with age might be explained by enhancement of suppressor T-cell activity rather than by a defect in B cells per se. Spleen cells from old mice were, therefore, treated with anti-Thy.1 serum and complement before transfer into irradiated young recipients together with SIII. Removal of T cells did not, however, restore the direct PFC responses of old cells toward young cells (Table 3). Taken together, these results pointed to a primary defect in antibody-forming-cell precursors ( AFCP) . G. SIII

Antigen-Binding

Cells in Old Mice

Despite the failure to find evidence of a reduction in total B-cell numbers, it was essential to test whether selective depletion of SIII AFCP had occurred with age. AFCP were quanitiated by radioautographic analysis of SIII antigen-binding cells (ABC). When the proportion of ABC in spleens from unimmunised old and young mice was compared, no difference was observed (Fig. 4), thereby excluding a gross defect in ABC. H.

Intensity

of SIII

Antigen-Binding

Cells in Old Mice

The finding of normal numbers of ABC to SIII in spleens from old mice did not exclude the possibility of changes in epitope density of receptor sites or reduced

R-CELL

ABC

IN DONOR

Young

FUNCTION

IN

OLD

ANTIBODY

SPLEENS

Old

RESPONSE

Young

FIG. 4. Quantitation of SIII antigen-binding The frequency of SIII ABC is correlated with same spleen cell suspensions following adolptive

33

MICE TO Sm

Old

cells in spleens from young and old mice. the antibody (direct PFC) response of the transfer into irradiated recipients.

avidity of antigen binding to them, either of which could lead to defective transformation of AFCP into antihocly-forming cells. The intensity of antigen binding was assessed by counting the number of silver grains present around each SIIIABC. Comparisons between young and old spleen cells were made from radioautographs of cell smears prepared under identical conditions and exposed for the same length of time. SIII-ABC were classified as l+, 2+, 3+, 4’. and 5+ according to the number of grains surrounding each cell, that is, 10-20, 20-30, 30-40, 40-50, and greater than 50, respectively. The percentage of ABC falling into each class was estimated for spleen cells ohtainecl from unimmunised mice and from mice 3 and 7 clays after challenge with 5 ,ug of SIII (Fig. 5). The data were subjected to a two-way analysis of variance from which the following conclusions have been drawn. First, there was no significant difference in intensity of antigen TABLE Effect of T-Cell

3

Depletion on the Adoptive Anti-S111 Antibody of Young and Old Spleen Cells

Source of donor spleen cells

Treatment of donor cells

Number of irradiated recipients

Young mice

Normal mouse serum plus complement Anti-Thy-l.2 serum plus complement Normal mouse serum plus complement Anti-Thy-l.2 serum plus complement

8

Old mice

a ‘Arithmetic

mean f. SE.

Response

Direct anti-S111 PFC/ spleen at 5 days

9,500 zt 1,800n

8

12,300 f

2,100

8

3,000 f

1,400

8

3,200 f

800

34

CALLARD,

80

I

BASTEN

AND

WATERS

OLD

1 Resting

v) 60 2 tfl

B B 9 g Y L

I

Resting

60.

Day 3

Day 7

40.

FIG. 5. Intensity of SIII antigen binding to spleen cells from young and old mice before immunisation (resting) and at 3 and 7 days after immunisation. Antigen-binding cells are classified according to the number of silver grains associated with each cell: + represents 10-20 grains pe,r cell ; f+ = 21-30; +++ = 31-40; ++++ = 41-50 ; and +++++ > 50.

binding to AFCP obtained from naive young and old mice. Second, immunisation was accompanied by a significant shift toward low intensity binding cells in both young and old mice (P < 0.001). This indicates a preferential loss of high intensity binding cells following immunisation due probably to transformation into non-antigen-binding antibody-forming cells. Third, the shift toward low intensity binding cells following immunisation was more pronounced in young mice than in old (P < 0.05), which is consistent with a reduction with age in the transformation potential of AFCP. DISCUSSION The experiments described in the previous paper (1) indicated that the loss of T-cell function with age is due to a qualitative abnormality in the T cells themselves. The aim of the present study was to test whether the reduced level of humoral immunity reported in old mice (2, 3) is due to a similar mechanism in B cells. This was particularly important since the loss of antibody-forming potential with age has been attributed to diminished B-cell numbers (21). B-cell function was assessed by measuring the mitogenic response to LPS and antibody production to the T-cell-independent antigen, SIII. The reduced level of 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 SIII (Figs. 1 and 3) were similar for old and young cells. Furthermore, a role for extracellular (environmental) influences was excluded by showing that the poor antibody-forming potential of old cells was still apparent after acloptive transfer into young irradiated recipients (Table 2).

B-CELL

FUNCTION

IN

OLD

MICE

3.5

A primary abnormality in B cells themselves was confirmed by showing that fewer spleen cells from old mice were capable of entering mitosis on stimulation with LPS (Fig. 2). This observation does not, however, distinguish between a reduction in the actual number of available B cells and a qualitative defect preventing the cells from responding to the mitogenic signal. A similar problem in interpretation is encountered when evaluating the results of the limiting dilution assay of Albright and Makinodan (22) and Price and Makinodan (21) which cannot be taken as definitive evidence for a quantitative loss of B cells with age. Indeed, when the number of B cells was measured directly in the current study by counting Ig- and FcR-bearing cells! an increase rather than a decrease was found in both spleen and lymph nodes from old mice (Table 1). It might, therefore, seem reasonable to conclude that the reduced LPS response in old mice is due to a qualitative abnormality in B cells. Since, however, only 20-30s of spleen cells respond to LPS, a reduction in a subpopulation of LPS-reactive B cells cannot be excluded. More definitive proof of a qualitative rather than quantitative abnormality in B cells was sought by analysing the antibody response of old mice to SIII in relation to the number of specific antigen-binding cells (ABC). Although antibody production to SIII does not require T-cell help (6), Baker et al. (20) have demonstrated the existence of T-cell-dependent regulatory influences in immune responses to SIII. An increase in suppressor T-cell activity with age was excluded here by showing that pretreatment of old spleens cells with anti-Thy.1 serum and complement did not enhance their antibody forming capacity to SIII in adoptive transfer (Table 2.) A loss of macrophage function is also considered to be an unlikely explanation for the poor responses of old mice to SIII since (i) antibody formation to thymusindependent antigens at least in vitro appears to be macrophage independent (23) ; and (ii) macrophages from old mice can support in e~ify~ antibody responses as effectively as those from young mice (24). Thus, the most reasonable interpretation of the data available points to a qualitative defect in B cells per se in old animals. The importance of using an assay system, the cellular components of which are well defined in cellular terms, is illustrated by the studies of Gerbasse-De Lima et al. (25). They compared the response of old and young mice to LPS-coated mouse red cells and found a reduction with age. This, however, could have been due to reduced helper T-cell rather than B-cell activity since other polysaccharide antigens such as SIII are rendered thymus-dependent when coated onto sheep red cells (26). The precise nature of the reduction in B-cell responsiveness to SIII in old mice was investigated by estimating the number of specific ABC before and after immunisation with SIII. The validity of this approach was based on previous observations which have indicated that ABC are B cells (27) and are the specific precursors of antibody-forming cells (28). The failure to detect a reduction in SIII-specific ABC in spleen cells from unprimed old mice (Fig. 4) argues strongly in favour of a qualitative, not a quantitative abnormality in B-cell function as the explanation of the loss of humoral immunity with age. Consistent with this interIn-etation were the studies of intensity of antigen binding to B cells (Fig. 5). 111 these, a 1)referential loss of high intensity binders was observed following imiininisation with SIII, which \vas more pronounced with cells from young mice than

36

CALLARD,

RASTEN

AND

WATERS

from old. In other words, more avid binding of antigen to B cells from old mice was required for transformation into antibody-formin, (+ cells. The refractoriness of old cells is, therefore, best explained by a membrane or intracellular defect resulting in a lower probability of triggering in response to a particular antigenic (or mitogenie) signal. ACKNOWLEDGMENTS This work of Australia

was supported by grants from the National Health and Medical and the University of Sydney Cancer Research Fund,

Research

Council

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28.

Callard, R., and Basten, A., Cell. Znwnunol. 31, 13, 1977. Makinodan, T., and Peterson, W. J., J. Zmnzz~nol. 93, 856, 1964. Makinodan, T., and Peterson, W. J., Deveolfi. Biol. 14, 96, 1966. Miller, J. F. A. P., Basten, A., Sprent, J., and Cheers, C., Cell. Zmmz~nol. 2, 469, 1971. Basten, A., and Mitchell, J. In “Immunobiology of the Macrophage” (D. Nelson, Ed.), Academic Press, New York, 1976. Basten, A., and Howard, J. G. In. “Contempory Topics in Immunobiology” (Davies and Carter, Eds.), Vol. 2, p. 265. Plenum Publishing Corp., New York, 1973. von Boehmer, H., and Shortman, K., J. Znznzzknol. Methods 2, 293, 1973. Heidelberger, M., Kendall, F. E., and Scherp, H. W., J. Exp. Med., 64, 559, 1936. Siskind, G. W., Paul, W. E., and Benacerraf, B., Zmnzztrrochenzistry 4, 455, 1967. Miller, J. F. A. P., and Warner, N. L., Znt. Arch. Allergy 40, 59, 1971. Greenwood, F. C., Hunter, W. M., and Glover, J. S., Biochem. J. 89, 114, 1963. Basten, A., Miller, J. F. A. P., Sprent, J., and Pye, J., J. Exp. Med. 135, 610, 1972. Byrt, P., and Ada, G. L., Zmmz~rzology 17, 503, 1969. Cunningham, A. J., and Szenberg, A., Znzmunology 14, 599, 1968. Askonas, B. A., Farthing, C. P., and Humphrey, J. H., Zwwkkkrkology 3, 336, 1960. Gottleib, C. F., J. Zmntunol. 113, 51, 1974. Snedecor, G. W., and Coch.ran, W. G., “Statistical Methods,” 6th ed. Iowa State University Press, Ames, Iowa, 1967. Taylor, E. W., In. “Drugs and the Cell Cycle” (A. M. Zimmerman, G. M. Padilla, and I. L. Cameron, Eds.), pp. 11-21. Academic Press, New York, 1973. Forni, L., and Pernis, B. Zn “Membrane Receptors of Lymphocytes” (M. Seligmann, J. L. Preud’homme, and F. M. Kourilsky, Eds.), p. 193. North-Holland, Amsterdam, 1975. Baker, P. J., Stashak, P. W., Amsbaugh, D. F., and Prescott, B., J. Znznzzknol. 112, 404, 1974. Price, G. B., and Makinodan, T., J. Zmmunol. 108, 413, 1972. Albright, J. F., and Makinodan, T., J. Cell. Physiol. 67, 185, 1966. Mosier, D. E., Johnson, B. M., Paul, W. E., and McMaster, P. R. B., J. Exp. Med. 139, 1354, 1974. Heidrick, M. L., and Makinodan, T., J. Zmmunol. 111, 1502, 1973. Gerbase-De Lima, M., Wilkinson, J., Smith, G. S., and Walford, R. L., J. Gero&ol. 29, 261, 1974. Braley-Mullen, H. C., J. Znamunol. 113, 1909, 1974. Paul, W. E., and Davie, J. M. In “Progress in Immunology” (B. Amos, Ed.), p. 637. Academic Press, New York, 1971. Basten, A., Miller, J. F. A. P., Warner, N. L., and Pye, J., Nature Nezv Biol. 231, 104, 1971.