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Cellular aspects of tolerance
CELLULAR
12, 205-213
IMMUNOLOGY
(1974)
Cellular VI.
The
Effect
of Age
MICHIO
Aspects
of Tolerance
on Responsiveness and Tolerance of SJL Mice l, *
FUJIWARA
3 AND BERNHARD
CINADER
Institute of Immunology, Departments of Medical Genetics, Medical and Clinical Biochemistry, Medical Sciences Building, University Toronto, Ontario, Cafaada M5S lA8 Received
September
Induceability
Biophysics,
of Toronto,
4, 1973
New born and 3-week-old SJL mice but not 8-12-week-old animals could be rendered tolerant to rabbit y-globulin. Animals reconstituted with thymus cells from 12-week-old donors and bone marrow cells from 3-week-old donors showed resistance to tolerance induction. Animals reconstituted with bone marrow cells from 12-weekold animals and thymus cells from 3-week-old donors could be rendered tolerant. Earlier work has shown that tolerance could be induced in older animals, if they were deprived of competent accessory cells. It was suggested that a lesion in the thymus cell population is expressed through the mediation of accessory cells. The possibility of a relation between resistance to tolerance induction and lymphoid malignancies was discussed.
INTRODUCTION In earlier papers of this series we have reported that the response of adult SJL mice to rabbit y-globulin (RGG) depended on cooperative participation of thymusderived (T) and accessory cells, and that B and T cells were involved in tolerance induction, that accessory cells tended to interfere with tolerance, and that this interference might be mediated through a thymus-derived cell type (1, 2). In this paper we shall deal with the effect of age on antibody responseand on tolerance induction. We shall provide experimental evidence that the age-dependent resistance to tolerance induction is, at least in part, attributable to a change in the cooperative capacity of T cells. 1 Supported by the Medical Research Council, the National Cancer Institute and the Ontario Heart Foundation. 2 Abbreviations : B cell : Bone marrow-derived progenitors of antibody-forming cells ; BM : Bone marrow cells; BM,: Bone marrow cells from normal donors ; BMt : Bone marrow cells from tolerant donors ; CFA : Complete Freund’s adjuvant ; GRBC: Goat erythrocytes; IFA : Incomplete Freund’s adjuvant ; PBS : Phosphate buffered saline ; PFC. : Indirect plaque-forming cells, assayed in agar; PFCr : Indirect plaque-forming cells, assayed in liquid; RGG-CFA : Rabbit y-globulin incorporated in ‘Freund’s complete adjuvant ; RGG-IFA : Rabbit r-globulin incorporated in Freund’s incomplete adjuvant; RGG: Rabbit y-globulin; Tt : Half-life of antigen elimination (in days) ; T cells : Thymus-derived cells ; Th : Thymus cells ; Th, : Thymus cells from normal donors; Tht : Thymus cells from tolerant donors. s Present address: Department of Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan. 205 Copyright All rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
206
FU JIWARA
MATERIALS
AND
CINADER
AND
METHODS
Inbred mice were purchased from Jackson Laboratory (Bar Harbor, ME). Hybrids were bred in our own colony from stock mice obtained from Jackson Laboratory. The strain designation J was common to all animals and was, therefore, omitted from designation in the text. Hybrids were identified by the name of the maternal animal, followed by the name of the paternal animal. Experimental mice were caged in groups of five and allowed free access to food and water. Composition of buffers, media (3), and adjuvants, procedures for purification of rabbit y-globulin (RGG ; 4)) iodination of RGG (5)) removal of aggregates from RGG and from its lightly iodinated derivative, preparation of heat-aggregated RGG (6)) covalent attachment of RGG to goat erythrocytes (GRBC; 7)) the agar(PFC,) and liquid- ( PFCr) hemolytic plaque assay (8-10)) tolerance induction, the elimination test, and the preparation of cell suspensions are given in preceding papers of this series (1, 2, 10). RESULTS Half-life of antigen elinaination as a function of age. There was an age-dependent change in the rate at which RGG was eliminated from the body of A and SJL mice (Table 1). In A mice, this change occurred between 6 and 8 weeks of age (p < 0.005) and there was no further change in the following 7 weeks of life. In SJL mice there was no age-dependent change between the ages of 6 and 11 weeks, but a decrease was seen in the half-life of l*lI-RGG by the 15th week of life ($ < 0.005) ; no corresponding changes were observed in Balb/c mice. Age and strain dependent diferences in the nztmber of plaque-faming cells. On immunization with aggregated RGG, SJL animals of all ages made a much larger indirect plaque-forming response (p < 0.005) than did A animals (Fig. 1). In A animals the PFCr increased 5.1 fold from the 3rd to the 5th week of life and 1.6 fold from the 5th to 8th week. In SJL mice the corresponding changes were 1.7 and 1.4 fold. The changes in PFC, for A were 4.4 and 2.0 and for SJL were 2.5 and TABLE HALF-LIFE
OF RGG
ELIMINATION
I AS A FUNCTION
Strain
Age in weeks 6
A
14.1 f
0.6
(5) BALB/c
Tt (4
SJL
OF AGE”
9.9 f
1.5
(3) 8.7 f 1.2 (10)
8
11
15
10.9 f 1.3 (14)
11.2 f 1.6 (36)
10.1 f 0.7 (10)
9.6 f
9.7 f
9.5 f
1.8
05) 8.6 31 1.6 (14)
1.9
(30) 7.8 f
2.1
(23) 1.0
6.5 f
(20)
D Animals were injected with 10 pg ‘311-RGG at the indicated age and the eliminated between the day of injection and the eighth day afterward. b Half-life in days. c (n) = number of animals in the group.
1.1
(8) half-life
of antigen
AGE
AND
IMMUNOLOGICAL
RESPONSIVENESS
207
1.9 fold. The ratio PFCr/PFC, for animals at 3 weeks, 5 weeks, and 8 weeks were 7.9, 9.1, and 3.1, respectively; for SJL animals of corresponding ages it was 10.1, 7.1, and 5.3, respectively. The relative changes in PFCi/PFC, ratio may be taken as an indication of age-dependent affinity maturation, which is similar in animals of both strains. The involvement of B and T cells in the age-dependent change in plaque-forming response. In order to determine the relative role of T and B cells in modifying the responsiveness with age, we reconstituted S-weeks-old irradiated (950 rad, 3 days before cell transfer) female SJL mice with lo8 thymus and 2 X IO7 bone marrow cells from 3-week and 12-week-old donors. Seven days later, these primary recipients becamespleen cell donors for lethally irradiated (850 rad, 2 hr before cell transfer) secondary recipients. These were injected twice with aggregated RGG (0.5 mg, iv, on the day cell transfer and ip 10 days later) and were sacrificed 4 days after the last injection (Fig. 2). As will be seen from Fig. 2, the reconstitution with bone marrow cells from 12-week-old donors causes only a marginal increase in plaque numbers [ThsBMa compared with ThsBMrz (0.1 > p > O.OS)], while the transfer of thymus cells from 12-week-old donors led to a 4-5-fold increase in plaque numbers (ThsBMs compared with ThrzBMs:p < 0.005). Neonatal induction of tolerawe in SJL mice. Newborn SJL mice, in different groups, were given either a single injection of 0.5 mg or three injections at 4%hr intervals of a total of 1.5 mg or eight injections at 4%hr intervals of a total of 4.0 mg of aggregate free RGG. In all cases the first injection was given within 15 hr of birth. At the age of 5 weeks the mice were immunized SCwith 0.25 mg
Age in weeks
1. The plaque-forming response as a function of age. A and SJL female mice were injected twice with 0.5 mg aggregated RGG. The first injection (iv) was given at the age in weeks shown on the horizontal axis; the second injection (ip) was given 10 days later. Animals were sacrificed 4 days after the last injection. The number of indirect plaque-forming cells was determined and is shown by the height of vertical bars. PFCI (height of dotted bar) and PFC. (height of hatched bar) were determined with spleen cells. Vertical lines indicate 21 standard deviation. FIG.
208
FU JIWARA
AND
CINADER
FIG. 2. Reconstitution of S-week-old SJL mice with thymus (Th) and bone marrow (BM) cells from 3-week and 12-week-old donors. Eight-week-old recipients were reconstituted 3 days after total body irradiation (9.50 rad). The age of the cell donor is given as a suffix. Spleen cells were transferred to secondary recipients which were immunized. The number of indirect plaque-forming cells is shown by the height of the bars.
RGG-CFA, and a week later 10 pg 1311-RGGwere injected and elimination of the antigen was followed. As can be seen from Fig. 3, unresponsiveness was induced and the extent of unresponsivenesswas dose related. The tolerant state after neonatal administration of 4 mg seemednearly complete if subsequent immunization was carried out with incomplete rather than complete Freund’s adjuvant. Male SJL mice were given neonatally 4 mg of aggregate free RGG in eight injections; they were immunized, when 5 weeks old, with RGG-IFA. 1311-RGGwas 9-
87-
6-
::“,
5-
E Iw 7 : =
4-
3-
2-
l-
0.5 Total dose (mg) inteded
1.5
4 .o
aggregate-free RGG into new born ankmals
FIG. 3. Half-life of ‘*‘I-RGG elimination from immunized of the neonatally injected dose of aggregate free RGG.
6-week-old
SJL mice as a function
AGE
AND
IMMUNOLOGICAL
Tht BM,
RESPONSIVENESS
Th, BMt
209
Th, EM,
FIG. 4. Reconstitution of lethally irradiated SJL mice with thymus and bone marrow cells from neonatally tolerized (Tht and BMt cells) and with cells from normal donors (Th, and BM, cells). Plaque numbers are designated by vertical bars (PFC,: height of hatched bar; PFC,: height of dotted bar).
a week later and the half-life of antigen elimination was found to be 12.9 f 0.4 days. It was, therefore, apparent that SJL mice could be rendered tolerant by neonatally administered antigen and that immunological responsivenessof T and B cells could be investigated with adequately tolerized SJL mice. SJL mice were injected with 4 mg aggregated free RGG, equally distributed into eight daily injections, the first being given within 15 hr of birth. When females were 5 weeks old they served as donors of Th, and BMt. Six-week-old females served as donors of Th, and BM,. Recipients were S-week-old lethally irradiated (8.50 rad) females who received iv lo8 thymus and 2 X 10’ bone marrow cells of the type shown on the horizontal axis of Fig. 4. Immediately afterward they were given iv 0.5 mg aggregrated RGG. The same dose was given ip 10 days later. Animals were sacrificed 4 days after the last injection and the number of PFC was determined. As can be seen from Fig. 4, animals reconstituted with thymus cells from neonatally tolerized donors were completely unresponsive ; animals reconstituted with bone marrow cells from tolerant donors were more responsive but still less responsive than were animals which had received bone marrow cells from donors which had not been injected neonatally. Age- and strain-dependent half-life difference; animals first rendered tolerant arrd then imwmnized. Animals, first rendered tolerant and then immunized with RGG, showed a much more continuous age-dependent decrease in half-life than did animals of the same strain which had been left untreated. This change, normalized to that of untreated animals, was more pronounced in Balb/c than in A mice but was most dramatic in SJL mice (Table 2). Attempts to imj&cate accessory cells in age-dependent changes to tolerance iwduceability. As judged by the half-life of 13’1-RGG elimination (T,) , S-week-old hybrids between A and SJL animals (A X SJL) were intermediate between A (T, = 9.1 -+ 1.5) and SJL (T+ = 1.7 2 0.7) mice (T, = 5.7 + 2.4) in their resistance to tolerance induction, whereas 5-week-old hybrids (T+ = 11.0 * 1.5) resemble their A parent (T+ = 10.3 -t 0.7) and not their SJL parent (Tt = 3.0 -+ injected
210
FU JIWARA
AND
TABLE TOLERANCE
INDUCTION
CINADER
2
AS A FUNCTION
Strain
AGI:~
OF
Age of mice in weeksb 3
5
8
12
12.7 f 1.0 (9) 0.90 * 0.08
10.3 f 0.7 (9) 0.95 f 0.13
9.1 f 1.5 (30) 0.81 f 0.18
8.4 f 0.9 (8) 0.83 f 0.11
(17) 0.97 f 0.20
8.2 f 2.2 (27) 0.85 f 0.28
7.2 f 1.6 (13) 0.74 f 0.23
3.0 f 2.5 (14) 0.35 f 0.30
1.7 f 0.7 (15) 0.22 f 0.09
0.4 f 0.3 (10) 0.06 f 0.05
A
Tt” w Ratio”
BALBjc
Ti
-
(4 Ratio
-
9.2
6.7 f 0.8
SJL
(6)
Ratio
0.77 f 0.14
f
0.8
a Animals were injected ip with aggregate free RGG (0.2 mg/of body weight), 2 weeks later they were given RGGIFA (0.25 mg, SC). One week after this injection, mI-RGG 10 pg was administered ip and the half-life of elimination was determined. * Age at which animals were given aggregate free RGG; elimination tests were always carried out 3 weeks after this injection. c Half-life in days. d Number of animals in the group. e Ratio = (half-life of mI-RGG elimination from the body of animals injected with aggregate free RGG))/(half-life of animals nof injected with aggregate free RGG).
2.5). We injected 5-week-old A X SJL hybrids iv with 1 x lo8 spleen cells from A or from SJL donors which had been irradiated (950 rad), 2 hr before they became donors. The recipients were concurrently given 5 mg aggregate free RGG and were immunized 2 weeks later with RGG-IFA. The half-life of 1311-RGG in normal animals was 13.1 -I- 2.3 days; in tolerized, immunized animals Ti was 11.8 +- 3.1 days; in tolerized, immunized SJL spleen cell recipients Tt was 12.3 + 2.1 ; and in tolerized, immunized A spleen cell recipients T* was 11.7 * 1.7 days. Thus, passive transfer of a source of accessory 4 or SJL cells did not affect the sensitivity to tolerance induction of 5-week-old animals. The role of thymus-derived cells in age-dependent changes in tolerance induceability. Tolerance was induced in S-week-old lethally irradiated mice which had been reconstituted with thymus and bone marrow cells from 3- or 12-week-old donors. The responsivenessof the spleen cells was determined after transfer to a secondary
lethally
irradiated
host
which
was
immunized.
The
indirect
plaque-
forming responseof this host was considerably greater (- X 5) when the primary host had received 12-week-old thymus cells than when it received thymus cell from a 3-week-old donor. However, the response was the same if the numbers of plaque-forming cells were normalized to those of corresponding animals reconstituted with cells from normal donors (Table 3). DISCUSSION The responsivenessto aggregated KGG of both A and SJL animals increases with age. The relative increase in plaque response from the third to the eighth
+ +
12
3
+ + + +
(in weeks)
cells
~~Y,o,’
Thymus
Comparison land3 1 and 5 3 and 5
cells
O.l>
PFCl of groups p >o.os p <0.005 p
+ +
12
(in weeks)
marrow
between
+ +
+ +
Age of donor 3
Bone
host
2and4 2 and 6 4and6
+ + +
-
Aggregate free RGG
p
ff f f
0.8 fh 29.0
host
0.2 6.1 > 28.1 1.4
0.5 2.0 >
PFCJlOG spleen cell
Secondary
CELLS
0.8 35.4 160.9 4.2
MARROW
p >0.4 p <0.005
4 5 3 4 3 4
Number of recipients
IN SJL MICE RECONSTITUTED WITH THYMUS AND BONE FROM 3-WEEK AND 12-WEEK-OLD DONORS~
Primary
INDUCTION
3
f
f
38.4 *
44.3
36.3
Ratiob
14.4
11.42
22.8
a Primary recipients were irradiated (9.50 rad) 3 days before the cell transplants were given. Tolerance was examined in secondary recipients which were reconstituted with spleen cells from the primary recipients. * Ratio between plaque numbers in secondary hosts when the primary host was not injected with aggregate free RGG and when the primary host was injected with aggregate free RGG.
1 2 3 4 5 6
Group
TOLERANCE
TABLE
212
FUJIWARA
AND
CINADER
week is greater in A than in SJL mice, but the absolute indirect plaque response of 3-week-old S JL mice is already as great as that of 8-week-old A mice (Fig. 2). From reconstitution experiments it became apparent that thymus cells from old mice, but not bone marrow cells, could confer on a recipient the greater responsiveness of the older animal. It is clear that we must normalize responses of tolerant animals to those of corresponding normal animals in any comparisons of responsiveness at different age ; this has been done wherever appropriate. Newborn SJL mice could be readily rendered tolerant and, even when they had become 6-weeks-old, the responsiveness was still reduced (Fig. 3). At the age of 3 weeks, SJL mice could acquire a degree of unresponsiveness which was comparable with that induced in A and Balb/c mice (Table 3). The resistance to tolerance induction was a gradually acquired modification of responsiveness. Such a modification might affect the rate of antigen elimination from normal animals directly and tolerance induceability indirectly. That this was not the case could be seen from normalized half-lives (ratios in Table 2). We attempted to identify the cellular basis of this change. We relied on reconstitution experiments in which the donors of thymus and bone marrow cells were either 3 or 12 weeks old (Fig. 2). Animals, reconstituted with thymus cells from normal old donors and with bone marrow cells from the normal young donors, gave a much higher response than did animals in which the ages of the donors were reversed. Thus there appeared to occur a thymus cell-dependent hyper-responsiveness with advancing age. Reconstitution experiments with cells from tolerant animals which were 3 and 12 weeks old showed that it was the thymus cell and not the bone marrow cell of the old animal which resulted in an incomplete state of tolerance (Table 3). If the responses of animals, reconstituted with tolerant cells, were normalized for the with cells from donors which responses of corresponding animals, reconstituted had not been rendered tolerant, there was no difference between animals reconstituted with thymus cells from old and with thymus cells from young animals. The failure to detect a difference might be connected with the high standard deviation of the normalized response of animals (Table 3). Thus, we could link the thymus cell with the age-dependent resistance to tolerance induction but could not safely interpret it as a consequence of heightened responsiveness to antigen. We have found that passive transfer of accessory cells does not transfer the age-dependent change in tolerance induceability. However, in earlier papers of this series (1, 2)) we have demonstrated that the removal of accessory cells permitted induction of tolerance in S-week-old animals. A synthesis of these apparently contradictory observations is possible. It seems conceivable that the accessory cell and its product act on the T cell, or that participation of the accessory cell is dependent on the T cell, that there is some age-dependent change in T cell function, and that this change results in hyper-responsiveness to antigen, possibly competitive advantage of the interaction which results in antibody formation rather than in tolerance induction. Thus, the lesion would be in the thymus cell population but the manifestation would be dependent on the presence of accessory cells. It would be attractive to attribute the lesion to a progressive loss of inhibitory T cells (12-14) or to a disturbance in the equilibrium between inhibitory and cooperating T cells (14-18), but a decision on the validity of this proposal must await appropriate experimentation. There are striking similarities in the behavior of SJL and NZB mice (19-22) ; genetic experi-
AGE
AND
IMMUNOLOGICAL
213
RESPONSIVENESS
merits (SJL x NZB) might help to resolve the question of whether or not the lesions of these two mice are identical. Of the four mouse strains, SJL, NZB, Balb/c, and B/W, which have proven relatively resistant to tolerance induction, all but B/W are predisposed to Hodgkin’slike reticulum cell neoplasm (23--3S), lymphomas (26)) or plasmacytomas (27), The alteration in immunological mechanism, which we have analysed in the preceding paper, may involve failure of T cell function which may be responsible for the development of lymphoid malignancies. Indeed, it is quite possible that the same virus may play a part in the development of the immunological abnormality and, directly or indirectly, in the development of neoplastic change (28-30). ACKNOWLEDGMENTS for
We are grateful to L. Horvath and Mrs. Sui Koh for technical his help in computing standard deviations of ratios.
assistance
and to P. K. Chung
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. 29. 30.
Fujiwara, M., and Cinader, B., Cell. Immunol. 12, 11, 1974. Fujiwara, M., and Cinader, B., Cell. Imnmnol. 12, 194, 1974. Parker, R. C., Castor, L. N., and McCulloch, E. A., 411~. N.Y. Acud. Sci. 5, 303, 1957. Fujiwara, M., Jap. J. Exp. Med. 41, 59, 1971. Sonoda, S., and Schlamowitz, M., Immu!lochemi.rfry 7, 885, 1970. Chiller, J. M., Habicht, G. S., and Weigle, W. O., Science 171, 813, 1971. Kaplan, A. M., and Freeman, M. J., P,roc. Sac. Esp. Biol. Med. 127, 574, 1968. Jerne, N. K., Nordin, A. A., and Henry, C., In “Conference on Cell-Bound Antibody,” Washington, D.C. (B. Amos and H. Koprowski, Eds.), p. 109. Wistar Institute Press, Philadelphia, 1963. Cunningham, A. J., and Szenberg, A., Immunology 14, 599, 1968. Kaplan, A. M., and Cinader, B., Cell. Immn~ol. 6, 429, 1973. Kaplan, A. M., and Cinader, B., Cell. Immznlol. 6, 442, 1973. Gershon, R. K., and Kondo, K., Immunology 21, 903, 1971. Jacobson, E. B., Herzenberg, L. A., Riblet, R., and Herzenberg, L. A., J. Exg. Med. 135, 1163, 1972. Cinader, B., Koh, S. W., and Kuksin, P., Cell. Zmmunol. 11, 170, 1974. Dutton, R. W., Falkoff, R., Hirst, J. A., Hoffmann, M., Kappler, J. W., Kettman, J. Ft., Lesley, J. F., and Vann, D., In “Progress in Immunology.” Proceedings of the 1st International Congress of Immunology, Washington, D.C., 1971 (B. Amos, Ed.), p. 355. New York, Academic Press, 1971. Sjiiberg, O., Andersson, J., and Miiller, G., J. Imnaunol. 109, 1379, 1972. Blaese, R. M., Martinez, C., and Good, R. A., J. Exp. Med. 119, 211, 1964. Lapp, W. S., and MGller, G., Immunology 17, 339, 1969. Staples, P. J., Steinberg, A. D., and Talal, N., J. Exp. Med. 131, 1223, 1970. Playfair, J. H. L., Immunology 21, 1073, 1971. Chused, T. M., Steinberg, A. D., and Parker, L. M., J. Zmtn~rrol. Ill, 52, 1973. Steinberg, A. D. Private communication. Murphy, E. D., J. Nat. Cawcer Inst. (USA) 42, 797, 1969. McIntire, K. R., and Law, L. W., J. Nat. Cancer Itzsf. (USA) 39, 1197, 1967. Wanebo, H. J., Gallmeier, W. M., Boyse, E. A., and Old, L. J., Science (Washington) 154, 901, 1966. Mellors, R. C., Blood 27, 435, 1966. Potter, M., and Robertson, C. L., 1. Nat. Cuncev Inst. (USA) 25, 847, 1960. Mellors, R. C., and Huang, C. Y., J. Exp. Med. 126, 53, 1967. Thivolet, J., Merrier, J. C.. Ruel, J. P., and Richard, M. H., Nrrfzrve (London) 214, 1134, 1967. Oldstone, M. B. A., Tishon, A., Chiller, J. M., Weigle, W. O., and Dixon, F., J. Immunol. 110, 1268, 1973.
12, 205-213
IMMUNOLOGY
(1974)
Cellular VI.
The
Effect
of Age
MICHIO
Aspects
of Tolerance
on Responsiveness and Tolerance of SJL Mice l, *
FUJIWARA
3 AND BERNHARD
CINADER
Institute of Immunology, Departments of Medical Genetics, Medical and Clinical Biochemistry, Medical Sciences Building, University Toronto, Ontario, Cafaada M5S lA8 Received
September
Induceability
Biophysics,
of Toronto,
4, 1973
New born and 3-week-old SJL mice but not 8-12-week-old animals could be rendered tolerant to rabbit y-globulin. Animals reconstituted with thymus cells from 12-week-old donors and bone marrow cells from 3-week-old donors showed resistance to tolerance induction. Animals reconstituted with bone marrow cells from 12-weekold animals and thymus cells from 3-week-old donors could be rendered tolerant. Earlier work has shown that tolerance could be induced in older animals, if they were deprived of competent accessory cells. It was suggested that a lesion in the thymus cell population is expressed through the mediation of accessory cells. The possibility of a relation between resistance to tolerance induction and lymphoid malignancies was discussed.
INTRODUCTION In earlier papers of this series we have reported that the response of adult SJL mice to rabbit y-globulin (RGG) depended on cooperative participation of thymusderived (T) and accessory cells, and that B and T cells were involved in tolerance induction, that accessory cells tended to interfere with tolerance, and that this interference might be mediated through a thymus-derived cell type (1, 2). In this paper we shall deal with the effect of age on antibody responseand on tolerance induction. We shall provide experimental evidence that the age-dependent resistance to tolerance induction is, at least in part, attributable to a change in the cooperative capacity of T cells. 1 Supported by the Medical Research Council, the National Cancer Institute and the Ontario Heart Foundation. 2 Abbreviations : B cell : Bone marrow-derived progenitors of antibody-forming cells ; BM : Bone marrow cells; BM,: Bone marrow cells from normal donors ; BMt : Bone marrow cells from tolerant donors ; CFA : Complete Freund’s adjuvant ; GRBC: Goat erythrocytes; IFA : Incomplete Freund’s adjuvant ; PBS : Phosphate buffered saline ; PFC. : Indirect plaque-forming cells, assayed in agar; PFCr : Indirect plaque-forming cells, assayed in liquid; RGG-CFA : Rabbit y-globulin incorporated in ‘Freund’s complete adjuvant ; RGG-IFA : Rabbit r-globulin incorporated in Freund’s incomplete adjuvant; RGG: Rabbit y-globulin; Tt : Half-life of antigen elimination (in days) ; T cells : Thymus-derived cells ; Th : Thymus cells ; Th, : Thymus cells from normal donors; Tht : Thymus cells from tolerant donors. s Present address: Department of Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan. 205 Copyright All rights
0 1974 by Academic Press, of reproduction in any form
Inc. reserved.
206
FU JIWARA
MATERIALS
AND
CINADER
AND
METHODS
Inbred mice were purchased from Jackson Laboratory (Bar Harbor, ME). Hybrids were bred in our own colony from stock mice obtained from Jackson Laboratory. The strain designation J was common to all animals and was, therefore, omitted from designation in the text. Hybrids were identified by the name of the maternal animal, followed by the name of the paternal animal. Experimental mice were caged in groups of five and allowed free access to food and water. Composition of buffers, media (3), and adjuvants, procedures for purification of rabbit y-globulin (RGG ; 4)) iodination of RGG (5)) removal of aggregates from RGG and from its lightly iodinated derivative, preparation of heat-aggregated RGG (6)) covalent attachment of RGG to goat erythrocytes (GRBC; 7)) the agar(PFC,) and liquid- ( PFCr) hemolytic plaque assay (8-10)) tolerance induction, the elimination test, and the preparation of cell suspensions are given in preceding papers of this series (1, 2, 10). RESULTS Half-life of antigen elinaination as a function of age. There was an age-dependent change in the rate at which RGG was eliminated from the body of A and SJL mice (Table 1). In A mice, this change occurred between 6 and 8 weeks of age (p < 0.005) and there was no further change in the following 7 weeks of life. In SJL mice there was no age-dependent change between the ages of 6 and 11 weeks, but a decrease was seen in the half-life of l*lI-RGG by the 15th week of life ($ < 0.005) ; no corresponding changes were observed in Balb/c mice. Age and strain dependent diferences in the nztmber of plaque-faming cells. On immunization with aggregated RGG, SJL animals of all ages made a much larger indirect plaque-forming response (p < 0.005) than did A animals (Fig. 1). In A animals the PFCr increased 5.1 fold from the 3rd to the 5th week of life and 1.6 fold from the 5th to 8th week. In SJL mice the corresponding changes were 1.7 and 1.4 fold. The changes in PFC, for A were 4.4 and 2.0 and for SJL were 2.5 and TABLE HALF-LIFE
OF RGG
ELIMINATION
I AS A FUNCTION
Strain
Age in weeks 6
A
14.1 f
0.6
(5) BALB/c
Tt (4
SJL
OF AGE”
9.9 f
1.5
(3) 8.7 f 1.2 (10)
8
11
15
10.9 f 1.3 (14)
11.2 f 1.6 (36)
10.1 f 0.7 (10)
9.6 f
9.7 f
9.5 f
1.8
05) 8.6 31 1.6 (14)
1.9
(30) 7.8 f
2.1
(23) 1.0
6.5 f
(20)
D Animals were injected with 10 pg ‘311-RGG at the indicated age and the eliminated between the day of injection and the eighth day afterward. b Half-life in days. c (n) = number of animals in the group.
1.1
(8) half-life
of antigen
AGE
AND
IMMUNOLOGICAL
RESPONSIVENESS
207
1.9 fold. The ratio PFCr/PFC, for animals at 3 weeks, 5 weeks, and 8 weeks were 7.9, 9.1, and 3.1, respectively; for SJL animals of corresponding ages it was 10.1, 7.1, and 5.3, respectively. The relative changes in PFCi/PFC, ratio may be taken as an indication of age-dependent affinity maturation, which is similar in animals of both strains. The involvement of B and T cells in the age-dependent change in plaque-forming response. In order to determine the relative role of T and B cells in modifying the responsiveness with age, we reconstituted S-weeks-old irradiated (950 rad, 3 days before cell transfer) female SJL mice with lo8 thymus and 2 X IO7 bone marrow cells from 3-week and 12-week-old donors. Seven days later, these primary recipients becamespleen cell donors for lethally irradiated (850 rad, 2 hr before cell transfer) secondary recipients. These were injected twice with aggregated RGG (0.5 mg, iv, on the day cell transfer and ip 10 days later) and were sacrificed 4 days after the last injection (Fig. 2). As will be seen from Fig. 2, the reconstitution with bone marrow cells from 12-week-old donors causes only a marginal increase in plaque numbers [ThsBMa compared with ThsBMrz (0.1 > p > O.OS)], while the transfer of thymus cells from 12-week-old donors led to a 4-5-fold increase in plaque numbers (ThsBMs compared with ThrzBMs:p < 0.005). Neonatal induction of tolerawe in SJL mice. Newborn SJL mice, in different groups, were given either a single injection of 0.5 mg or three injections at 4%hr intervals of a total of 1.5 mg or eight injections at 4%hr intervals of a total of 4.0 mg of aggregate free RGG. In all cases the first injection was given within 15 hr of birth. At the age of 5 weeks the mice were immunized SCwith 0.25 mg
Age in weeks
1. The plaque-forming response as a function of age. A and SJL female mice were injected twice with 0.5 mg aggregated RGG. The first injection (iv) was given at the age in weeks shown on the horizontal axis; the second injection (ip) was given 10 days later. Animals were sacrificed 4 days after the last injection. The number of indirect plaque-forming cells was determined and is shown by the height of vertical bars. PFCI (height of dotted bar) and PFC. (height of hatched bar) were determined with spleen cells. Vertical lines indicate 21 standard deviation. FIG.
208
FU JIWARA
AND
CINADER
FIG. 2. Reconstitution of S-week-old SJL mice with thymus (Th) and bone marrow (BM) cells from 3-week and 12-week-old donors. Eight-week-old recipients were reconstituted 3 days after total body irradiation (9.50 rad). The age of the cell donor is given as a suffix. Spleen cells were transferred to secondary recipients which were immunized. The number of indirect plaque-forming cells is shown by the height of the bars.
RGG-CFA, and a week later 10 pg 1311-RGGwere injected and elimination of the antigen was followed. As can be seen from Fig. 3, unresponsiveness was induced and the extent of unresponsivenesswas dose related. The tolerant state after neonatal administration of 4 mg seemednearly complete if subsequent immunization was carried out with incomplete rather than complete Freund’s adjuvant. Male SJL mice were given neonatally 4 mg of aggregate free RGG in eight injections; they were immunized, when 5 weeks old, with RGG-IFA. 1311-RGGwas 9-
87-
6-
::“,
5-
E Iw 7 : =
4-
3-
2-
l-
0.5 Total dose (mg) inteded
1.5
4 .o
aggregate-free RGG into new born ankmals
FIG. 3. Half-life of ‘*‘I-RGG elimination from immunized of the neonatally injected dose of aggregate free RGG.
6-week-old
SJL mice as a function
AGE
AND
IMMUNOLOGICAL
Tht BM,
RESPONSIVENESS
Th, BMt
209
Th, EM,
FIG. 4. Reconstitution of lethally irradiated SJL mice with thymus and bone marrow cells from neonatally tolerized (Tht and BMt cells) and with cells from normal donors (Th, and BM, cells). Plaque numbers are designated by vertical bars (PFC,: height of hatched bar; PFC,: height of dotted bar).
a week later and the half-life of antigen elimination was found to be 12.9 f 0.4 days. It was, therefore, apparent that SJL mice could be rendered tolerant by neonatally administered antigen and that immunological responsivenessof T and B cells could be investigated with adequately tolerized SJL mice. SJL mice were injected with 4 mg aggregated free RGG, equally distributed into eight daily injections, the first being given within 15 hr of birth. When females were 5 weeks old they served as donors of Th, and BMt. Six-week-old females served as donors of Th, and BM,. Recipients were S-week-old lethally irradiated (8.50 rad) females who received iv lo8 thymus and 2 X 10’ bone marrow cells of the type shown on the horizontal axis of Fig. 4. Immediately afterward they were given iv 0.5 mg aggregrated RGG. The same dose was given ip 10 days later. Animals were sacrificed 4 days after the last injection and the number of PFC was determined. As can be seen from Fig. 4, animals reconstituted with thymus cells from neonatally tolerized donors were completely unresponsive ; animals reconstituted with bone marrow cells from tolerant donors were more responsive but still less responsive than were animals which had received bone marrow cells from donors which had not been injected neonatally. Age- and strain-dependent half-life difference; animals first rendered tolerant arrd then imwmnized. Animals, first rendered tolerant and then immunized with RGG, showed a much more continuous age-dependent decrease in half-life than did animals of the same strain which had been left untreated. This change, normalized to that of untreated animals, was more pronounced in Balb/c than in A mice but was most dramatic in SJL mice (Table 2). Attempts to imj&cate accessory cells in age-dependent changes to tolerance iwduceability. As judged by the half-life of 13’1-RGG elimination (T,) , S-week-old hybrids between A and SJL animals (A X SJL) were intermediate between A (T, = 9.1 -+ 1.5) and SJL (T+ = 1.7 2 0.7) mice (T, = 5.7 + 2.4) in their resistance to tolerance induction, whereas 5-week-old hybrids (T+ = 11.0 * 1.5) resemble their A parent (T+ = 10.3 -t 0.7) and not their SJL parent (Tt = 3.0 -+ injected
210
FU JIWARA
AND
TABLE TOLERANCE
INDUCTION
CINADER
2
AS A FUNCTION
Strain
AGI:~
OF
Age of mice in weeksb 3
5
8
12
12.7 f 1.0 (9) 0.90 * 0.08
10.3 f 0.7 (9) 0.95 f 0.13
9.1 f 1.5 (30) 0.81 f 0.18
8.4 f 0.9 (8) 0.83 f 0.11
(17) 0.97 f 0.20
8.2 f 2.2 (27) 0.85 f 0.28
7.2 f 1.6 (13) 0.74 f 0.23
3.0 f 2.5 (14) 0.35 f 0.30
1.7 f 0.7 (15) 0.22 f 0.09
0.4 f 0.3 (10) 0.06 f 0.05
A
Tt” w Ratio”
BALBjc
Ti
-
(4 Ratio
-
9.2
6.7 f 0.8
SJL
(6)
Ratio
0.77 f 0.14
f
0.8
a Animals were injected ip with aggregate free RGG (0.2 mg/of body weight), 2 weeks later they were given RGGIFA (0.25 mg, SC). One week after this injection, mI-RGG 10 pg was administered ip and the half-life of elimination was determined. * Age at which animals were given aggregate free RGG; elimination tests were always carried out 3 weeks after this injection. c Half-life in days. d Number of animals in the group. e Ratio = (half-life of mI-RGG elimination from the body of animals injected with aggregate free RGG))/(half-life of animals nof injected with aggregate free RGG).
2.5). We injected 5-week-old A X SJL hybrids iv with 1 x lo8 spleen cells from A or from SJL donors which had been irradiated (950 rad), 2 hr before they became donors. The recipients were concurrently given 5 mg aggregate free RGG and were immunized 2 weeks later with RGG-IFA. The half-life of 1311-RGG in normal animals was 13.1 -I- 2.3 days; in tolerized, immunized animals Ti was 11.8 +- 3.1 days; in tolerized, immunized SJL spleen cell recipients Tt was 12.3 + 2.1 ; and in tolerized, immunized A spleen cell recipients T* was 11.7 * 1.7 days. Thus, passive transfer of a source of accessory 4 or SJL cells did not affect the sensitivity to tolerance induction of 5-week-old animals. The role of thymus-derived cells in age-dependent changes in tolerance induceability. Tolerance was induced in S-week-old lethally irradiated mice which had been reconstituted with thymus and bone marrow cells from 3- or 12-week-old donors. The responsivenessof the spleen cells was determined after transfer to a secondary
lethally
irradiated
host
which
was
immunized.
The
indirect
plaque-
forming responseof this host was considerably greater (- X 5) when the primary host had received 12-week-old thymus cells than when it received thymus cell from a 3-week-old donor. However, the response was the same if the numbers of plaque-forming cells were normalized to those of corresponding animals reconstituted with cells from normal donors (Table 3). DISCUSSION The responsivenessto aggregated KGG of both A and SJL animals increases with age. The relative increase in plaque response from the third to the eighth
+ +
12
3
+ + + +
(in weeks)
cells
~~Y,o,’
Thymus
Comparison land3 1 and 5 3 and 5
cells
O.l>
PFCl of groups p >o.os p <0.005 p
+ +
12
(in weeks)
marrow
between
+ +
+ +
Age of donor 3
Bone
host
2and4 2 and 6 4and6
+ + +
-
Aggregate free RGG
p
ff f f
0.8 fh 29.0
host
0.2 6.1 > 28.1 1.4
0.5 2.0 >
PFCJlOG spleen cell
Secondary
CELLS
0.8 35.4 160.9 4.2
MARROW
p >0.4 p <0.005
4 5 3 4 3 4
Number of recipients
IN SJL MICE RECONSTITUTED WITH THYMUS AND BONE FROM 3-WEEK AND 12-WEEK-OLD DONORS~
Primary
INDUCTION
3
f
f
38.4 *
44.3
36.3
Ratiob
14.4
11.42
22.8
a Primary recipients were irradiated (9.50 rad) 3 days before the cell transplants were given. Tolerance was examined in secondary recipients which were reconstituted with spleen cells from the primary recipients. * Ratio between plaque numbers in secondary hosts when the primary host was not injected with aggregate free RGG and when the primary host was injected with aggregate free RGG.
1 2 3 4 5 6
Group
TOLERANCE
TABLE
212
FUJIWARA
AND
CINADER
week is greater in A than in SJL mice, but the absolute indirect plaque response of 3-week-old S JL mice is already as great as that of 8-week-old A mice (Fig. 2). From reconstitution experiments it became apparent that thymus cells from old mice, but not bone marrow cells, could confer on a recipient the greater responsiveness of the older animal. It is clear that we must normalize responses of tolerant animals to those of corresponding normal animals in any comparisons of responsiveness at different age ; this has been done wherever appropriate. Newborn SJL mice could be readily rendered tolerant and, even when they had become 6-weeks-old, the responsiveness was still reduced (Fig. 3). At the age of 3 weeks, SJL mice could acquire a degree of unresponsiveness which was comparable with that induced in A and Balb/c mice (Table 3). The resistance to tolerance induction was a gradually acquired modification of responsiveness. Such a modification might affect the rate of antigen elimination from normal animals directly and tolerance induceability indirectly. That this was not the case could be seen from normalized half-lives (ratios in Table 2). We attempted to identify the cellular basis of this change. We relied on reconstitution experiments in which the donors of thymus and bone marrow cells were either 3 or 12 weeks old (Fig. 2). Animals, reconstituted with thymus cells from normal old donors and with bone marrow cells from the normal young donors, gave a much higher response than did animals in which the ages of the donors were reversed. Thus there appeared to occur a thymus cell-dependent hyper-responsiveness with advancing age. Reconstitution experiments with cells from tolerant animals which were 3 and 12 weeks old showed that it was the thymus cell and not the bone marrow cell of the old animal which resulted in an incomplete state of tolerance (Table 3). If the responses of animals, reconstituted with tolerant cells, were normalized for the with cells from donors which responses of corresponding animals, reconstituted had not been rendered tolerant, there was no difference between animals reconstituted with thymus cells from old and with thymus cells from young animals. The failure to detect a difference might be connected with the high standard deviation of the normalized response of animals (Table 3). Thus, we could link the thymus cell with the age-dependent resistance to tolerance induction but could not safely interpret it as a consequence of heightened responsiveness to antigen. We have found that passive transfer of accessory cells does not transfer the age-dependent change in tolerance induceability. However, in earlier papers of this series (1, 2)) we have demonstrated that the removal of accessory cells permitted induction of tolerance in S-week-old animals. A synthesis of these apparently contradictory observations is possible. It seems conceivable that the accessory cell and its product act on the T cell, or that participation of the accessory cell is dependent on the T cell, that there is some age-dependent change in T cell function, and that this change results in hyper-responsiveness to antigen, possibly competitive advantage of the interaction which results in antibody formation rather than in tolerance induction. Thus, the lesion would be in the thymus cell population but the manifestation would be dependent on the presence of accessory cells. It would be attractive to attribute the lesion to a progressive loss of inhibitory T cells (12-14) or to a disturbance in the equilibrium between inhibitory and cooperating T cells (14-18), but a decision on the validity of this proposal must await appropriate experimentation. There are striking similarities in the behavior of SJL and NZB mice (19-22) ; genetic experi-
AGE
AND
IMMUNOLOGICAL
213
RESPONSIVENESS
merits (SJL x NZB) might help to resolve the question of whether or not the lesions of these two mice are identical. Of the four mouse strains, SJL, NZB, Balb/c, and B/W, which have proven relatively resistant to tolerance induction, all but B/W are predisposed to Hodgkin’slike reticulum cell neoplasm (23--3S), lymphomas (26)) or plasmacytomas (27), The alteration in immunological mechanism, which we have analysed in the preceding paper, may involve failure of T cell function which may be responsible for the development of lymphoid malignancies. Indeed, it is quite possible that the same virus may play a part in the development of the immunological abnormality and, directly or indirectly, in the development of neoplastic change (28-30). ACKNOWLEDGMENTS for
We are grateful to L. Horvath and Mrs. Sui Koh for technical his help in computing standard deviations of ratios.
assistance
and to P. K. Chung
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