Phenotypic and functional properties of B lymphocytes from aged mice

Mechanisms ofAgeins and Development, 51 (1990) 223--241

223

Elsevier Scientific Pubfishers Ireland Ltd.

PHENOTYPIC AND FUNCTIONAL PROPERTIES OF B LYMPHOCYTES FROM AGED MICE*

BONDADA SUBBARAOa'**'t, JOAN MORRIS" and R.J.KRYSCIO b •Department o f Microbiology and Immunology and Sanders-Brown Center on Aging, bDepartment o f Statistics, University o f Kentucky, Lexington, KY, 40536-0230 (U.S.A.)

(Received March 25th, 1989)

SUMMARY

Phenotypic and functional properties of B lymphocytes from individual young and old mice of different inbred strains were studied. B lymphocyte subpopulations defined by the ratios of the densities of cell surface IgM and lgD were found to be ~tered with age. However, such alterations in B cell subsets were found only in '30--40~/0 of the old mice. B cell mitogenic responses to anti-~ and anti-Lyb2 antibodies were decreased in a majority of DBAJ2 mice. Proliferative responses to LPS and anti-t~ were reduced only in a minority of CBA/Ca mice but there was a very good correlation in the responsiveness of the old mice to LPS and anti-/~. The anomalous properties of the individual old mice of these inbred strains may be due to a heterogeneity in the effects of aging or due to environmental influences.

Key words: B lymphocytes, Inbred strains of mice, Lymphocyte surface molecules, Lymphocyte activation, Aging, Immune function. INTRODUCTION

Immune responses to a number of antigenic stimuli are profoundly altered with increasing age. Numerous investigations have documented the central role of T lymphocyes in the age associated immune abnormalities [1]. Although the phenotypic analyses of B lymphocytes failed to reveal any changes in B cells in aged animals, several studies demonstrated intrinsic B cell deficiencies associated *This work was supported in part by the grants AI21490, AG05731 and the PSP funds of the University of Kentucky: **SB is a recipient of a career research development award (AG00422). t T o whom correspondence should be addressed at: Room 205, Sanders-Brown Building, University of Kentucky, Lexington, KY 40536-0230, U.S.A. 0047-6374/90/$03.50 Printed and Published in Ireland

© 1990 Elsevier Scientific Publishers lreland Ltd.

224 with increasing age [2--12]. Many of these studies employed thymic independent (TI) antigens, presumably to directly measure B cell functions [3,6--10]. Interestingly, responses to TI antigens of type I are not found to be very much affected by age, though some evidence to the contrary exists [6,7,13,14]. In general, immune responses to TI antigens of the type 2 [14] such as TNP-Ficoll, pneumococoal polysaccharide etc., are decreased in old animals [3,8,9]. Therefore, it is conceivable that the selective deficiencies in responses to the type 2 TI antigens may be due to alterations in the relative distribution of B cell subpopulations in old mice. At the phenotypic level B cell subsets are defined either by the presence or the absence of the differentiation antigens Lyb 3, 5 and 7 or by the ratio of the densities of cell surface IgM and IgD [15,16]. With regard to the surface Ig levels, there are three subpopulations of B lymphocytes in which population I expresses more IgD than IgM, population II has equivalent amounts of IgM and IgD on the B cell surface and population III has less IgD than IgM on the B cell membrane [16]. Further, population I is absent in CBA/N mice, which have an X-linked immune deficiency and in neonatal mice, resulting in the inability of B cells from both mice to respond to a challenge with the type 2 TI antigens [15--17]. The relative proportions of these B lymphocyte subpopulations in individual young and old mice, of different inbred strains were examined by dual color immunofluorescence analysis. At the functional level, the ability of B cells from old and young mice to respond to stimulation with anti-IgM and anti-Lyb2 antibodies were studied [19]. We find that there are profound differences in the composition of B cell subsets between young and old mice but that individual old mice differ from each other in the relative content of these B cell subpopulations. Also, proliferative responses to stimulation with anti-/a and anti°Lyb2 antibodies were defective in some old mice, though the deficiency was more pronounced in the old mice of the DBA/2 strain. MATERIALS AND METHODS

Mice Young (2--4 months of age) and old CBA/Ca, DBA/2 (22--24 months of age) and (C57BL/6 × DBA/2)F~ mice (28 months of age) were obtained from the National Institute on Aging. In some of the experiments, young mice were obtained from the Jackson laboratory (Bar Harbor, ME). The aged mice were carefully examined for the presence of any tumors or splenomegaly and any mice with tumors or enlarged spleen were excluded from the study.

Reagents Affinity purified anti-~ and monoclonal anti-Lyb2 antibody from the clone 10.1.D2 were prepared as described before [19,20]. These antibodies were dig-

225 ested with pepsin to generate (Fab)2 fragments which were purified as reported earlier [21]. Monoclonal anti-thyl (clone 13-4), rat anti-Lyl and rat anti-mouse Lyt2 (clones 53--7,53--6) antibodies were respectively used as ascites and culture supernatants [22,23]. Fluoresceinated avidin and phycoerythrin conjugated avidin were purchased from Becton Dickinson monoclonal center (Mountain View, CA) and Biomeda Corp. (Foster City, CA). Fluoresceinated (Fab)2 anti-/~ was purchased from Southern Biotechnology Associates, Inc. (Birmingham, AL). Goat antibody to Y2 heavy chains was affinity purified from immune goat sera and was conjugated with fluorescein isothiocyanate in our laboratory. Monoclonal anti-Ia antibodies were derived from the clones MKD6(IAd), 10.3.6.2 (IAk) and 14.4.4(IE d.k) [24--26].

Preparation of lymphocytes and cell culture Spleens were removed aseptically and a single cell suspension was prepared. The spleen cells were washed with Hank's Balanced salt solution (HBSS) several times. For functional responses, 2.5 X 105 spleen cells were cultured in triplicate in 0.2 ml of Iscove's F12 medium supplemented with 5°?o fetal calf serum. They were stimulated with 50 pg/ml lipopolysaccharide or affinity purified goat anti-/~ or monoclonal antibody to the Lyb2 surface antigen for 2 days and the cell proliferation was assessed by a 4.0 h pulse with 1.0 pCi of pH]thymidine (spec. act. 2 Ci/mMol, New England Nuclear, Boston, MA) and harvesting the cultures onto glass fiber filters with a Skatron automatic cell harvester. The incorporated radioactivity was measured with a Packard liquid scintillation counter and the results were expressed as geometric mean of triplicates along with the standard error. For depletion of T cells, spleen cells were incubated with anti-thyl.2, anti-Lyl and anti-Lyt2 antibodies for 45 rain on ice. Then the spleen cells were washed once and were incubated with mouse anti-rat kappa antibody and Low Tox M rabbit complement (Accurate chem. Sci. Corp., Westbury, NY) at 37oc for 30 min and were washed three times before use.

Immunofluorescence analysis Freshly isolated spleen cells were washed and resuspended in sorter buffer, which consisted of HBSS without phenol red, supplemented with 0.1070 bovine serum albumin and 0.1 °7o sodium azide. Ceils were stained with optimal concentrations of the monoclonal antibodies (10.3.6.2 for IA k, MKD6 for IAd and 14.4.4 for IE d,k and 10.4.22 for IgD) for 30 min at 4°C and washed twice with sorter buffer. They were then stained with fluoresceinated goat anti-IgG2 antibody for 30 min at 4°C and washed twice with sorter buffer. The cells stained with FITC conjugated antibodies were incubated with propidium iodide which would be taken up only by dead cells. Becton Dickinson FACS 420 or FACSTAR machines with the Argon laser were employed for flow cytometric analysis.

226 Forward light scatter was employed to gate on the lymphocytes. Nonviable lymphocytes were excluded by virtue of their red fluorescence. Green fluorescence of the stained cells was analyzed using logarithmic amplification. Fluorescence positive cells were defined by comparing with the profiles of the unstained cells and of the cells stained only with the second step antibody. To measure the relative amounts of IgM and IgD expressed on B cells, spleen cells were stained sequentially with FITC (Fab) 2 goat anti-/~, unconjugated IgG 2 myeloma protein, Biotin conjugated monoclonal anti-Y2 antibody and phycorythrin conjugated avidin. Green fluorescence, which is a measure of the IgM density, and red fluorescence, which measures the amount of IgD, were measured using 488 nM line of the Argon laser and employing 530/30 and 575/26 filters respectively for green and red fluorescence emissions. Fluorescence measurements were made on 10 000 cells and a contour plot of green versus red fluorescence was generated by the Consort 30 software. These contour plots are essentially cross-sections of isometric representations (3-dimensional) of the frequency of cells, green and red fluorescence, such that each cross section contains 4, 8, 16, 32 and 64 or more cells with the given values in the red and green fluorescence channels. The three B cell populations with different ratios of IgM and IgD were defined by comparing the profiles to those of B cells from CBA/N mice which lack population I B cells [13,15--17]. In each experiment, the fluorescence limits for the three B cell populations were defined for the young mice and the same conditions were employed for the old mice. Statistics Arithmetic means and standard deviations were calculated for each group and strain of mouse, as well as for all young and old mice. To compare the mean values of young and old mice, the hypothesis that the variances of these two populations were sufficiently alike was evaluated by the Snedecor's F-test. The variance ratio, F, and the confidence limits were calculated using an in house basic program and ABSTAT statistical package on an IBM PS2/50 computer. Whenever the variances were alike, the statistical significance of the differences in the group means was evaluated by the Student's t-test. Since the Ftest assumes that the data is normally distributed, the phenotypic properties of young and old mice of each strain were subjected to the Shapiro-Wilk test for normality [27]. The hypothesis that the variances of these two populations were equal was tested by Snedecor's F-test and by the Ansari-Bradley test in those cases where the data was not normally distributed [28]. The hypothesis that the mean response of these two populations were equal was tested by using an approximate student's t-test based on Satterwhaite's method because unequal variances were consistently encountered [28]. In those cases where non-normal data was obtained, the distribution of responses were compared between populations by using the Wilcoxon rank sum test. Finally, the predictive value of

227 changes in each parameter, on the remaining properties of old mice was evaluated by calculating the chi-square coefficient and the probability using the ABSTAT statistical package (Anderson-Bell Corp., Parker, Co.). RESULTS

B cell subpopulations defined by the expression of surface lgM and IgD on B cells from young and old mice The numbers of the IgM ÷ IgD ÷ B cells and the surface densities of these membrane immunoglobulin isotypes were determined by flow cytometry. The total numbers of Ig÷ cells were uniform in old mice. However, we detected a large variation among individual old mice when the relative ratios of IgM and IgD were measured. Therefore, we surveyed a large number of old mice of two different inbred strains, and one F 1 hybrid, on individual basis for both phenotypic and functional properties. The average numbers of IgM÷ IgD ÷ B cells were very similar in young and old mice (Table I) which is in agreement with previous studies [4,29]. Also, there was no strain dependent variation. Next, the distribution of B cell subpopulations defined by the relative amounts of surffice IgM and IgD were examined. Typical two dimensional plots of such data and the definition of the three subpopulations in young and old mice are shown in Fig. 1. Summary data for all the three groups of mice studied is presented in Table II. As noted in Table II, we found that in five out of nine cases the data for old mice exhibited non-normai distribution. However, all the values for the B cell subsets in young mice and the results for the remaining four cases of old mice were normally distributed. Therefore, as described in the methods, appropriate statistical tests TABLE I IgM ° IgD ÷ CELLS IN Y O U N G A N D O L D MICE"

Strain

Youn~

%IgM÷IglT spleen cells ~ CBA/Ca 42.89 :t: 7.64 (19) DBA/2 51.72 -',- 6.81 All mice d

Old

P-value"

46.98 .¢- 9.61 (49) 49.88 -4- 13.24

<0.01

(18)

(50)

48.09 :t: 8.04 (45)

47.40 :t: 11.63 (116)

<0.56 <0.71

•The n u m b e r o f IgM* IgD* cells in the spleen were determined by flow cytome~ry as described in the methods. bThe values represent arithmetic m e a n 4- standard deviation. 'The numbers in the parentheses refer to the total n u m b e r o f mice o f each strain studied. dThese include C B A / C a , D B A / 2 , a n d (C57BL/6 × DBA/2)Ft mice. "The m e a n values for y o n n g and old mice were compared by Students t-test.

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TABLE II B CELL SUBPOPULATIONS IN YOUNG AND OLD MICE" Strain

CBA/Ca CBA/Ca DBA/2 DBA/2 (BL/6 x DBA/2)F~ (BL/6 x DBA/2)FI

Age group ~

% of splenic B cell~.d Population I I&D > I&M

Population II 18l) • IsM

Population I!I 18D < IgM

Young (13) Old

46.38 ± 6.23 46.51

39.92 ± 4.29 36.77

(39)

± 11.06

± 15,38

Young (15) Old

52.33 ± 8.16 44.17

38.10 ± 9.75 43.42*

(43)

± 16.46

± 14.33

Young (6) Old (12)

51.17 ± 4.67 50.0 ± 17.89

35.33 ± 4.61 35.58* ± 13.94

13.69 ± 2.87 16.09" ± 12.78 9.43 ± 3.32 12.44" ± 8.56 15.33 ± 1.37 14.58" ± 5.44

oSubpopulations were measured as shown in Fig. I. bNumbers of mice studied in each age group are shown in the parentheses. cValues represent arithmetic mean + S.D. of each population expressed as % of total IgM+ lgD+ cells in the spleen. The values with star as superscript are derived from populations that were nonnormal. In such cases, nonparametric methods as described in the Methods section, were employed to compare means and variances. ~l'he hypothesis that the variances of means for young and old populations were the same was tested by the variance ratio (F) test. The F-values for each B cell subpopulation in young and old mice of any one particular strain ranged from a low of 2.064 to a high of 13.34. Considering the high number of degrees of freedom the P-values ranged from 0.0000 to 0.0712 rejecting the hypothesis that the variances were alike.

(e.g. S t u d e n t ' s t-test versus a n a p p r o x i m a t e s t u d e n t ' s t-test o r t h e W i l c o x o n test) were e m p l o y e d t o c o m p a r e t h e m e a n values o f each B cell s u b p o p u l a t i o n . I n all b u t o n e s i t u a t i o n , t h e r e was n o statistically significant d i f f e r e n c e b e t w e e n t h e n u m b e r s o f B cells in each subset in y o u n g a n d o l d mice. H o w e v e r , f o r t h e D B A / 2 s t r a i n o f mice, t h e r e was a statistically significant ( P < 0.02) decrease in t h e p o p u l a t i o n I B cells in t h e o l d mice. E v e n t h o u g h m o s t o f t h e m e a n values were n o t s i g n i f i c a n t l y d i f f e r e n t in y o u n g a n d o l d mice, t h e r e a p p e a r e d t o b e g r e a t e r i n d i v i d u a l v a r i a t i o n in o l d mice as seen b y the d o u b l i n g o f the s t a n d a r d d e v i a t i o n . T h e e q u a l i t y o f t h e v a r i a n c e for these p o p u l a t i o n s ( y o u n g a n d old) o f mice was e v a l u a t e d b y t h e F - t e s t a n d t h e F - v a l u e s suggested t h a t the v a r i a n c e s were n o t e q u a l . O n c e a g a i n , n o n p a r a m e t r i c statistical m e t h o d s (e.g. A n s a r i B r a d l e y test) were e m p l o y e d t o c o m p a r e t h e v a r i a n c e s o f g r o u p s t h a t d i d n o t satisfy the S h a p i r o - W i l k test o f n o r m a l i t y . Since such tests v a l i d a t e d the conclusions d e r i v e d f r o m the F - t e s t in every s i t u a t i o n , o n l y F - v a l u e s a r e p r o v i d e d in t h e r e m a i n d e r o f this s t u d y . T h e r e f o r e , t h e levels o f these B cell s u b p o p u l a t i o n s

230

B Cell Subsets In Young And Old Mice go O3

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Fig. 2. Distribution of B cell subpopulations in individual young and old mice. Analysis and definition of the B cell subpopulations were performed as described in the legend to Fig. 1 and in the methods section. Young and old mice of DBA/2, (C57BL/6 × DBA/2) F~ and CBA/Ca strains were studied. Results are compilation of data from 19 experiments.

TABLE III PHENOTYPIC ABNORMALITIES OF B CELLS FROM OLD MICE

Phenotype

No. o f mice with abnormal value~No, o f mice studied"

% Abnormal old mice

IgM < IgD b (population I) lgM ~ IgD (population II) lgM < IgD~ (populalon III)

47/99

47.47

39/99

39.39

33/99

33.3

•The old mice were all 19--22 months of age except the B6D2 F I hybrids which were 28 months old. The data is a pool of 28 experiments. bpopulations I and II were analyzed as described in Table II and Fig. 1. The values were considered abnormal if they were ± 20% (two times the standard deviation) or more of the mean value for control young mice in the same experiment. cMice were taken as abnormal if the proportion of population III is ± 50% (three times the standard deviation) of that for young mice.

231 i n i n d i v i d u a l y o u n g a n d old mice are s h o w n as scatter d i a g r a m s i n Fig. 2. T o o b t a i n a n idea o f the f r e q u e n c y o f the old mice with a b n o r m a l n u m b e r s o f these B cell s u b p o p u l a t i o n s , i n d i v i d u a l old mice were classified as a b n o r m a l if the % o f B cells i n each s u b p o p u l a t i o n differed f r o m the m e a n value for y o u n g mice (studied i n the same experiment) b y two s t a n d a r d deviations or more. These frequencies were s u m m a r i z e d i n T a b l e III. Such a n analysis d e m o n s t r a t e d t h a t B cells f r o m old mice were f r e q u e n t l y a b n o r m a l b y these criteria. T h e frequencies o f the a b n o r m a l mice were n e a r l y the same in all the strains studied. However, the changes i n the d i s t r i b u t i o n o f B cell subsets were n o t u n i f o r m l y low or high a n d hence the differences in the m e a n values for y o u n g a n d old mice were n o t statistically significant.

Distribution o f la antigens on B cells and the levels o f Ia* B cells in old mice B lymphocytes express class II molecules (Ia antigens) and the expression of the Ia antigens is enhanced during the early stages of B cell activation [30]. Since the enhanced expression of Ia is apparently related to B cell function, the

TABLE IV EFFECT OF AGE ON THE la* CELLS AND Ia ANTIGEN DENSITY" Mice ~

% la* cells

Young DBA/2 (18)

59.72 ± 6.76

Old DBA/2 (53)

60.11 ± 11.59 51.90 ± 4.67

Young CBA/Ca (21) Old CBA/Ca (69) All mice young (53)

54.80 ± 14.22 58.56 ± 8.04

All mice old (139)

57.39 ± 14.11

F-value ¢

2.6668 (P -- 0.0124)

8.9803 (P = 0.000)

3.0465 (P = 0.000)

la density d (% o f young)

% Abnormal mice"

112.0 ±40.3

34.6

104.5 ±31.5

26.1

110.10

28.5

±46.1

'Results are expressed as arithmetic mean ± S.D. The statistical significance of the differences between young and old mice was evaluated by the Student's t-test and the P-values are provided. bNumbers of mice in each age group were given in the parentheses. erhe F-values were determined by the Snedecor's variance ratio test. The values in parentheses represent the probabilities. ~Fhe Ia density for old mouse B cells was expressed as % control of the value for young mice used in the same experiment. el'he old mice were designated as abnormal if the Ia density was ± 40% (2 × S.D.) or more of the mean la density on B cells from young mice studied in the same experiment.

232

numbers of Ia ÷ cells in the spleen and the densities of Ia antigens on B cells were quantitated by flow cytometry and the results are summarized in Table IV. The F-test demonstrated that the variances for the young and old mice were not equivalent and that the older mice exhibited greater variation. The mean values were however nearly the same in all the mice. During the course of this study, we found that four old C B A / C a mice had very few if any Ia ÷ B cells in the spleen even though the n u m b e r of Ig ÷ ceils was nearly the same as that in young counterparts. One D B A / 2 and two B6D2F1 mice also had reduced, but detectable, numbers (20070) o f Ia ÷ spleen cells whereas the levels o f Ig ÷ spleen cells were near normal (40--50°7o). However, except for these 7 out o f 149 old mice studied, in all the remaining animals the numbers o f Ia ÷ cells correlated well with the n u m b e r o f Ig ÷ cells. The reason for this rare deficiency of Ia expression on Ig ÷ cells in old mice is not known. The Ia density, as measured by the mean fluorescence intensity, was expressed as °70 control o f the mean fluorescence value for control young mice in the same experiment. Once again, the average value for Ia density in old

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233

mice was similar to that of young mice, but the values for individual mice varied greatly (Table IV) and therefore the individual mouse data is shown in Fig. 3. Old mice were classified as abnormal if the mean fluorescence value was ± 40070 of the value for control young mice (equal to two times standard deviation) and this analysis shows that 28.5070 of the old mice have abnormal Ia phenotypes (Table IV). Further, there was a preponderance of mice with B cells that had increased cell surface Ia (28 mice) compared to those mice with B cells expressing less Ia (13 mice), than the young mice. Functional responses of B cells Finally to determine if the phenotypic abnormalities in the B cells resulted in any changes in the functional responses, proliferation of B cells to three mitogenie stimuli were evaluated for each individual old mouse. The proliferation inducing stimuli chosen were lipopolysaccharide, and antibodies to the B cell surface molecules, IgM and Lyb2. All of these agents have been shown to directly induce B cell mitogenesis [15,19,31]. Responses of individual mice, from one experiment, were shown in Table V. Once again, individual old mice responded differently, such that some animals had responses which were as good as the young mice, whereas others had increased or decreased responses (Fig. 4). Old mice were considered abnormal if the proliferative response was + 5007o of the mean value for young mice evaluated in the same experiment. The numbers of abnormal mice for each stimulus and the strain of mouse are presented in Table VI. It appears that only a small fraction (25.6¢/e) of old mice have altered proliferative response to LPS whereas a larger proportion of the old mice have altered responses to anti-~ (43.9070) and to anti-Lyb2 (64.7070). Interestingly, the abnormalities in LPS response represent both a group of mice (11 07o)with elevated response, and a group with decreased response (17.5070), giving rise to the total value of 28.5070 for the fraction of mice with altered response to

TABLE V P R O L I F E R A T I V E RESPONSES O F S P L E E N CELLS F R O M Y O U N G A N D O L D D B A / 2 MICE TO B C E L L MITOGENS" Mitogen ~

Young mice

Old Mice

1

2

1

2

3

4

5

6

7

8

116.0 82.5

66.6 25.9

80.5 74.4

83.1 53.9

91.3 80.7

189.2 0

64.1 40.0

44.2 69.0

90.8 68.4

Acpm x 10~/cttlture b LPS 108.2 Anti-~ 80.0

•The ages o f young a n d old D B A / 2 mice were respectively 3 and 22 m o n t h s . W h e proliferative responses in the absence o f any mitogen were subtracted from the experimental groups. The values represent m e a n s o f tripficate cultures. The values that were :t: 50% o f the m e a n response o f the young mice were underfined and such mice were classified as abnormal.

234

Proliferation Of B Cells From Old Mice To Stimulation With LPS, Anti-/~, And Anti-Lyb2 O

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.<: Fig. 4. Proliferation o f B cells from individual old mice to stimulation with anti-/a, LPS and monoclonal anti-Lyb2. Proliferative responses o f spleen cells from young a n d old mice were measured under identical conditions as described in the Methods. The responses o f each old mice were expressed as % control response compared to the m e a n response o f 2 - - 3 y o u n g mice for each mitogen. Results are compilation o f data from 19 experiments.

T A B L E VI S U M M A R Y OF T H E P R O L I F E R A T I V E RESPONSES OF Y O U N G A N D O L D M I C E '

Strain

DBA/2 CBA/Ca All mice studied (DBA/2, C B A / C a and B 6 D 2 F )

No. o f old mice

39 49 91

% Old mice with altered response LPS

anti-/a antibody

anti-Lyb2 antibody

28.2 32.7 30.7

64.1 24.4 43.9

64.7 ND 64.7

•Old mice were classified as abnormal if the spleen cell proliferative response was ± 50% o f the mean value for young mice. bThe data for anti-Lyb2 is derived only from D B A / 2 mice.

235

LPS. For anti-/~, a smaller group of old mice (80/0) exhibited enhanced responses whereas a majority (35°70) had lower than normal response. In the case of anti-Lyb2, only one out of 31 mice studied had a response higher than that of control young mice (Fig. 4). When the mitogenic responses of individual strains were examined, the incidence of abnormal mice was uniform for LPS in all the strain combinations studied. Old mice exhibited altered responses to anti-/~ more frequently in the DBA/2 strain of mice. The frequency of mice with deficient responses to antiLyb2 was as high as that for anti-~ in the DBA/2 mice. Responses to anti-Lyb2 were not evaluated in the CBA/Ca mice, because our monoclonal antibody to Lyb2 does not recognize the Lyb 2.2 allele of the CBA/Ca mice [15,19]. Since the proliferative responses to anti-/~ and anti-Lyb2 may also be affected by the binding of the stimulating antibody to the Fc receptors on B cells, we evaluated the responses of B cells to (Fab) 2 fragments of the antibodies. As seen from Table VII, the numbers of old mice with altered responses to these stimuli was not affected by the use of antibodies with or without Fc fragments for stimulating the B cells. Also, the addition of lymphokines, in the form of supernatant from activated antigen specific T cells, did not have any effect. However, depletion of T cells from the responding spleen cells increased the number of old mice with reduced responses to anti-/~ or anti-Lyb 2, suggesting that the B ceils from old mice were less able to respond to these stimuli in the absence of T cells.

Correlation of phenotype and functional responses It is clear that individual old mice exhibit unique phenotypes with respect to B cell surface properties and the functional responses. We questioned if changes in any one of the five parameters (B cell subpopulations, Ia density,

T A B L E VII I N F L U E N C E O F F R E C E P T O R S , T CELLS A N D L Y M P H O K I N E S ON B C E L L P R O L I F E R A T I V E RESPONSES OF O L D M I C E Source o f cells or addition to culture Untreated spleen T depleted spleen cells I (FAB)2 antibody ~ Intact antibody • Lymphokine a

% abnormal mice ~ Anti-la

anti-L y b 2

No. o f mice studied

52.9 72.7 47.8 42.6 55.6

47.1 82.3 57.1 66.6 ND

17 22 23 68 9

'Only D B A / 2 mice were studied extensively for the effect of T cells and addition o f lymphokines. bAbnormal mice were defined as described in Tables V and Vl. cData for (Fab)~ anti-Lyb2 was obtained with 7 D B A / 2 mice and 27 D B A / 2 mice for the intact antibody. dLymphokines were added in the form of supernatant from an antigen specific T cell line.

236 TABLEVIII CHI-SQUARETEST OF THE PREDICTIVENATUREOF THE PHENOTYPEAND FUNCTIONAL ABNORMALITIESIN OLD MICE" Chi-square/probabilitf (n) Parameter

Population I

LPS

anti-~

LPS

1.55/0.8171 (61) 7.56/0.1090 (61) 2.85/0.2410 (20) 5.32/0.2561 (58)

--

_

21.62/0.0002 (89) 3.57/0.1652 (33) 7.35/0.1184 (81)

--

_

15.24/0.0042 (32) 2.26/0.6883 (80)

--

antiola anti-Lyb2 Ia d e n s i t y

anti-Lyb2

1.26/0.5333 (26)

•Levels of the B cell subpopulation I (see Figs. 1, 2 and Table II) proliferative responses to LPS, anti-la, anti-Lyb2 and Ia density were chosen for comparison. Mice were classified as high, low or normal in comparisonto the mean responses of youngmicetested in the same experiment. q2orrelations were performed only if there was data for both the parameters in test. The numbers of old mice with meaningfuldata for each comparisonweregiven in the parentheses.

proliferative response to LPS, anti-/a and anti-Lyb2), we investigated, would be predictive of the pattern in the remaining parameters. For this purpose, old mice were classified as high, normal or low, depending on the measure o f that parameter being respectively higher than, equal to or lower than the mean values of 2 - - 4 control young mice ( ± 2 standard deviations) studied in the same experiment. Then these response patterns for all the old mice were cross correlated for each parameter by the chi-squared test, using a computer program. The values for the chi-square and the probabilities are summarized in Table VIII. Only three correlations appear to have any statistical significance. Thus, on an average, mice with altered response to LPS, also have altered response to anti-/a (P < 0.002). Similarly, changes in the responses to anti-la corresponded closely with the alterations in the responses to anti-Lyb2 (P < 0.02). At a slightly lower probability, there is a correlation in the mice that have modified levels of population I B cells and altered responses to anti-la or anti-Lyb2. This is not surprising, since both anti-/a and anti-Lyb2 have been suggested to stimulate proliferation o f a mature population o f B cells represented by the population I, which is absent from C B A / N mice and neonatal mice [15,17,19,31]. DISCUSSION Our studies demonstrated that B lymphocytes from some aged mice have defective phenotypic and functional properties. Interestingly, the B lymphocyte

237 abnormalities were expressed in an idiosyncratic manner, even though inbred strains of mice were being studied. Thus only 30 to 50~/0 of the mice had alterations in B cell subpopnlations, defined by the ratios of cell surface IgM and IgD, or in the ability of their B cells to proliferate in response to various mitogenlc stimuli. The heterogeneity was not associated with the presence of apparent tumors or splenomegaiy of any type, since all such mice were excluded from the current analysis. This is important, since, in a previous study a distinct group of old mice with splenomegaiy and lower B cell responses were identified [8]. For all hut one functional property, (anti-/~ response, see below) there was aiso no strain dependent variation since the fraction of mice with abnormal phenotypes was approximately same in two different mouse strains (DBA/2 and CBA/Ca) and in one hybrid strain between C57BL/6 and DBA/2. Finally, the variant phenotypes of the individual old mice were not due to further differences in the exact age since all DBA/2 and CBA/Ca mice studied were between 22 and 24 months old and the B6D2F~ mice were 28 months old. Importantly, the mice wRh anamolous B cell phenotypes or functional responses were found in a majority of the individual experiments (though the exact per cent of ahnormai mice was variable between experiments), even though the age group and the cohort status of the animals for any particular experiment was identical for all the mice. Our observations may provide explanations for the apparently contradictory findings on the effects of age on B cell function. Many studies that measure responses of individual animals or responses to type 2 TI antigens, such as pneumococcai polysaccharide and TNP-FicolI, appear to find consistent B cell deficiencies [3,8--10]. This is in agreement with our findings about a reduction of the mature B cells (population I) and responses to anti-/~ which behave like responses to type 2 TI antigen in their dependence on a mature B cell subset [15 --17,31]. The responses to LPS and other T dependent antigens, as well as antigen-specific precursors to some but not other antigens Sere found to be altered with age in some, but not all studies [2,4---12,14,32--34]. Many, if not all, of these studies appeared to have employed pooled B cells derived from several old mice and in such instances responsive B cells from normal old mice may dominate the response obscuring the defective B cells from abnormal old mice. So far, we do not have a method to phenotypicaily determine the functional status of an animal without sacrificing the animal and measuring several B cell phenotypes and properties. Conceivably, peripheral blood lymphocytes from these mice might exhibit the same properties and phenotyping such cells may provide the means to identify the old mice with aberrant B cells. The individual variation among the genetically homogeneous mice of various inbred strains is intriguing. One possible explanation is that these animals may have been differentially influenced by their environment resulting in defective B cells only in some of the mice. The old mice employed in these studies were all

238

aged in germ free colonies and mice in any single experiment had identical cohort status. They were housed under pathogen free conditions and were employed for the studies often within days after arrival, or rarely within a few weeks after arrival, into our pathogen free animal care facility. Therefore, mice in any given experiment would have been exposed to the same environmental stimuli. However, it is still conceivable that the environmental stimulation has influenced the status o f B cells in each mouse differently, such that only some individual old mice o f these inbred animals have B cell abnormalities. Alternatively, the heterogeneity is influenced by some epigenetic events, which contribute to the aging process in a multifactorial manner such that even members of an inbred strain age differently. Whatever may be the explanation, it is clear that the immunological phenotypes and the responses o f the aged mice are not uniform even among the members of a presumably genetically homogeneous population o f inbred animals. Such a heterogenerity in the aged mice might contribute to the variability observed by us. On the basis o f the variance ratios calculated for the F-test, it appears that 3 - 6 times as many aged mice as the young animals have to be studied on an individual basis to obtain statistically significant age associated differences. If the environmental influences are antigenic in nature, it is more likely that they would affect the mature B cells but not the bone marrow stem cells. On the otherhand, epigenetic events are more likely to influence both mature B cells and their Ig- B cell precursors. Since B cells are generated fresh from the bone marrow precursors throughout the life o f the animal, analysis of B cells generated by transfer o f bone marrow cells from abnormal old mice into irradiated recipients may distinguish between these two possibilities. Indeed, several such transfer studies have been performed previously and often the newly generated B cells were found to be relatively normal in their functional properties though a few contradictory results exist [12,14,35--38]. However, in these experiments, the nature o f the B cells in the bone marrow donor was not characterized and often bone marrow cells from several donors were pooled. Since we find that only 30--40°70 of the animals had defective B cells, any experiment employing pooled cells from several donors may not detect B cell abnormalities, since the normal cells may compete out the abnormal precursors. Indeed, such a competition was demonstrated by Francus et al. [39]. These authors found that bone marrow cells from old mice were as effective as those from young mice in repopulating irradiated recipients when the stem cells from young or old mice were transferred into separate recipients. However, when allotype marked bone marrow cells from young and old donors were transferred into the same recipient, there was a preferential repopulation by the bone marrow cells from the young donors [39]. Furthermore, recent studies with in vitro bone marrow cultures, demonstrated a quantitative defect in the ability of the stem cells from old mice to generate mature B cells [40,41]. However, only two characteristics, surface Ig expression and proliferative response to LPS were analyzed.

239

The defects in the B cell proliferation responses appear to be due to intrinsic defects in B cells, since the proportion of abnormal mice did not decrease when T cells were depleted or by the use of antibodies lacking Fc fragments to stimulate B cells. In fact, the proportion of mice with reduced responses to anti-~ but not LPS was increased when T cell depleted spleen cells were studied. This is consistent with the recent findings on the positive influence of T cell derived B cell growth factors on the B cell response to anti-p [42,43]. We found no strain specific variation for a majority of the B cell abnormalities. However, the proliferative responses of B cells in aged DBA/2 mice to anti-~ were reduced more often than in others. In these mice, the reduced proliferative response to anti-/~ correlated very well with a low response to antiLyb2 (P = 0.0004) and weakly with the reduced levels of population I B cells (P -- 0.0896) but not with the response to LPS (P = 0.8620). This result is consistent with the observation that in young mice of various strains, the B cells in the population I respond to anti-p better than the remaining B cells [17,31]. In addition, the same mature B cell population is thought to be involved in responses to anti-Lyb2, whereas all the B cells can proliferate equally well in response to LPS [6,1-5,19]. The numbers of CBA/Ca mice with low responses to anti-p were much smaller than those in the DBA/2 strain. Also, in CBA/Ca mice there was excellent correlation between altered responses to anti-~ and LPS which is reflected strongly in the correlation analysis of the responses of all the mice (Table VIII). This finding points to the need for a survey of more than one inbred mouse strain before the findings on aged mice can be generalized. The strain dependency appears to be related to the aging process and its effect on B cell function since, B cells from young DBA/2 mice respond to anti-/~ as well as the B cells from young CBA/Ca mice. In conclusion, we found that B cells from old mice exhibited age associated changes in phenotypes and functional properties. The B cell deficiencies were, however, idiosyncratic to each individual old mouse, even among members of inbred strains of mice. ACKNOWLEDGEMENT

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