- Email: [email protected]
Role of I-J in neonatal suppression
CELLULAR
IMMUNOLOGY
80, 267-278
(1983)
Role of I-J in Neonatal BERTIE F. ARGYRIS~ANDCARL
Suppression’ WALTENBAUGH
Department of Microbiology, Upstate Medical Center, Syracuse, New York 13210, and Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611 Received March 4, 1983; accepted May 17, 1983 Spleen cells from neonatal mice belonging to strains with the I-J-b or I-J-’ genotype, were treated with anti-I-Jb or anti-I-J’ antibody and complement. This reduces their suppressor cell activity as demonstrated by a decrease in the ability to suppress the mixed-lymphocyte reactivity of adult spleen cells. Injection of anti-I-J antibody into neonatal mice also reduces splenic suppressor cell activity prematurely. The removal of suppressor cells from neonatal spleen does not result in an immediate increase in mixed-lymphocyte reactivity (cell-mediated immunity) but does hasten the development of mixed-lymphocyte reactivity in the young mice. The results are discussed in light of the hypothesis that suppressor cells inhibit the function of immunocompetent cells in the neonatal mouse and control the development of immunocompetence.
INTRODUCTION Newborn mice are immunologically immature. The immunological immaturity has, at least in part, been contributed to high suppressor cell activity (l-4) and low macrophage function (5). The suppressor cell in the spleen of newborn mice has been shown to be an I-J+, Lyt-l+, Lyt-2- T cell (3, 4) but several investigators claim that neonatal splenic non-T cells also have suppressor cell activity (6-8). We have shown that the splenic suppressor cell activity in postnatal mice is inversely correlated with the degree of immunological maturity (2). We have presented evidence which suggests strongly that a gradual increase in the number and/or function of macrophages after birth results in a gradual decrease in suppressor cell activity (2, 5). We have postulated that this gradual decrease in suppressor cell activity allows for immunocompetence to emerge. In this paper we are testing this hypothesis. We have treated neonatal spleen cells with anti-I-J antibody and determined whether the resulting decrease in suppressor cell activity is accompanied by an increase in immunocompetence, as measured by mixed-lymphocyte reactivity. We have also injected neonatal mice with anti-I-J antibody to test whether this decreases suppressor cell activity and subsequently leads to earlier development of immunocompetence. ’ This work was supported by USPHS Research Grant CA 15462 from the National Cancer Institute and Grant AI 18072 from National Institutes of Health. ’ To whom requests for reprints should be addressed. 267 0008-8749/83
$3.00
Copyngbt 0 1983 by Academic Press, Inc. All rights of reproduction m any form reserved.
268
ARGYRIS
AND
MATERIALS
WALTENBAUGH
AND
METHODS
Mice. Adult C57BL/6, DBA/2, and adult B6D2F1 mice were purchased from Jackson Laboratories, Bar Harbor, Maine, as 7-week-old animals, and used at 12 weeks of age. Bl0.A (3R) and BIO.A (5R) were obtained as breeding pairs from Dr. Jack Stimpfling and Dr. Phillippa Marrack, respectively. They were bred by brother and sister matings in our laboratory. Neonatal mice were obtained from our own colony. BDF, mice are the Fi offspring from C57BL/6 female and DBA/2 male mice. Cell suspensions. Single-cell suspensions were prepared by pressing spleens through size 50 stainless-steel mesh into phosphate-buffered saline. Red blood cells, from adult spleens only, were lysed by hypotonic shock (9). Cells were counted in 0.5% acetic acid and suspended in RPM1 1640 medium (Gibco), supplemented with 25 mM Hepes, 10 pg/lOO ml streptomycin, 10,000 U/ 100 ml penicillin, and 5 mg/lOO ml gentamicin. Mixed-lymphocyte culture (MLC). Mixed-lymphocyte cultures were carried out as described previously (3) with 3 X lo5 spleen cells responding to 3 X 10’ DBA/2 stimulating spleen cells in a total volume of 0.2 ml tissue culture medium (RPMI) with 10% of fetal calf serum. Up to 100 X lo6 stimulating cells were pretreated with 50 kg mitomycin C (kindly donated to us by Dr. William Bradner, Bristol Laboratories, Syracuse, N.Y.), in 2 ml of phosphate-buffered saline, for 20 min in a 37°C shaking water bath. The mitomycin-treated cells were washed three times before use. This dose of mitomycin is adequate to prevent incorporation of [3H]TdR into lymphocytes. Cultures were labeled with 1 PCi of [3H]TdR (Amersham) after 72 hr and harvested with an automatic cell harvester (0. Hiller) after 96 hr. Uptake of [3H]TdR by the proliferating cells was determined in a Packard scintillation counter. Triplicate cultures of syngeneic (AA,) and allogeneic (AB,) cells were carried out. The stimulation index (SI) represents the ratio of average counts in AB, and AA,,, cultures. The percentage response of the adult responding cells (control) was arbitrarily set at 100%. The mixed-lymphocyte reactivity of neonatal responding spleen cells was compared to that of syngeneic adult responding spleen cells (donors 3-4 months of age) and expressed as percentage responsiveness by calculating the ratio of the differences in counts per minute between AB, and AA,,, cultures, e.g., % response =
AB, - AA,,, (neonatal) x loo AB, - AA,,, (adult) ’
where adult denotes the use of adult responding cells and neonatal denotes the use of neonatal responding cells in MLC. Measurement of suppressor cell activity. The suppressor cell activity of neonatal spleen cells was measured as described previously (3). In short, 5 and 2.5 X 10’ mitomycin-treated neonatal spleen cells were added to MLC assay cultures of 3 X 10’ adult syngeneic spleen cells responding to 3 X 10’ DBA/2 mitomycin-treated stimulating spleen cells. Previously we have shown that the suppressor cell activity of neonatal spleen cells is mitomycin resistant (1, 3). The control cultures received an equal number of mitomycin-treated spleen cells from adult mice syngeneic to the responding spleen cells. Mitomycin treatment of the added cells was carried out as described above for the stimulating spleen cells in MLC. The percentage suppression was calculated as follows
I-J AND NEONATAL
% suppression = 100 -
SUPPRESSION
269
AB, - AA, (neonatal) x 100 AB, - AA,,, (adult) I ’
where adult denotes cultures receiving added adult cells and neonatal denotes cultures to which neonatal cells were added. Production of anti-Z-J sera. “Conventional” anti-I-J sera were prepared by weekly ip injections of Bl0.A (3R) spleen cells into BIO.A (5R) mice for anti-I-Jb and in the reverse direction for anti-I-Jk. Fifty million spleen cells were injected each time. In most cases the mice started producing an effective antiserum after 10-l 1 weeks. Serum was collected by bleeding the mice from the orbital cavity. The sera were tested by measuring loss of suppressor activity of neonatal spleen cells (Bl0.A (3R) for a-I-Jb and BIO.A (5R) for a-I-Jk). Sera were stored in small aliquots at -6O“C. Production of monoclonal anti-Z-P. Monoclonal anti-I-J-b antibodies (WF 9.20.8) have been previously characterized ( 10). The antibodies were used as a culture fluid diluted in RPM1 medium. Treatment with anti-Z-J sera and complement. Thirty to fifty million neonatal spleen cells were incubated with a I: 10 dilution of the appropriate (Y-I-J serum for 1 hr at 4°C. The cells were centrifuged, resuspended in a 1: 10 dilution of rabbit complement (Low-Tox; Cedar Lanes), and reincubated for l/2 hr in a 37°C shaking water bath. Control tubes included neonatal cells treated with RPM1 or RPM1 and complement. The cells were washed three times. Half of the cell suspension was used for the measurement of mixed-lymphocyte reactivity (see above); the other half of the cell suspension was treated with mitomycin (see above) and used for the measurement of suppressor cell activity (see above). Injection of anti-Z-J antibody. Neonatal mice were injected ip with 0.1 ml of antibody via a dorsal approach. The injection site was sealed with a surgical spray to prevent leakage of the injected material. Three or four mice from each litter were injected with the same aliquot and their spleen cells pooled at the time of sacrifice. The neonates were marked by clipping the tail and/or right/left toe nails. The control mice were injected with phosphate-buffered saline only. After injection, the neonates were returned to their mothers and kept as an isolated litter until they were sacrificed. Spleen cells from each group were pooled and made into a single-cell suspension. One half of the neonatal cell suspension was treated with mitomycin (see above) and used for the measurement of suppressor cell activity (see above). The other half of the cell suspension was used to measure mixed-lymphocyte reactivity, using mitomycintreated DBA/2 spleen cells as stimulating spleen cells. Only the adult and not the neonatal spleen cells were subjected to hypotonic shock, for removal of red blood cells. Presentation of data. The tables contain representative data from one experiment. All experiments were repeated three to four times. Statistical analysis was carried out with the small-sample method of Fisher. RESULTS Previously (3) we have demonstrated that treatment of neonatal spleen cells with specific anti-I-J sera results in decreased suppressor cell activity. At that time we also showed that the effect was specific because BIO.A (3R) neonatal spleen cells (I-Jb)
270
ARGYRIS
AND
WALTENBAUGH
treated with anti-I-Jb serum and complement lost suppressor cell activity whereas treatment with anti-I-Jk had no effect (3). In the following experiments we determine whether this decrease in suppressor cell activity results in an increased mixed-lymphocyte reactivity. The data in Table 1 confirm our previous results (3) and indicate that the high suppressor celI activity (Table 1, line 4) in neonatal B 1O.A (5R) mice is decreased considerably after treating the neonatal spleen cells with anti-I-Jk serum and complement (Table 1, line 5). However, when we use these neonatal spleen cells as responder cells in mixed-lymphocyte cultures we do not see a significant change in percentage responsiveness (Table 1, lines 2 and 3). Similar data are presented in Table 2 where spleen cells from neonatal B1O.A (3R) mice were treated with specific anti-I-Jb serum and complement. The results indicate that suppressor cell activity is lost (Table 2, lines 5 and 6), but percentage responsiveness of mixed-lymphocyte reactivity does not rise significantly (Table 2, lines 2 and 3). The data in Tables 1 and 2 suggest that (a) suppressor cell activity and mixedlymphocyte reactivity are unrelated or (b) a time interval is required for the immunocompetent cells to escape from the suppressive influence of the suppressor cells. To distinguish between these two possibilities, we changed our methodology and injected the neonatal mice with anti-I-J antisera in order to follow the events taking place in vivo. In the experiment depicted in Table 3, we injected BIO.A (5R) mice ip on the frrst, second, and third days alter birth (days 1,2, and 3) with 0.1 ml of anti-I-Jk antiserum and tested the splenic suppressor cell activity 3 days later (Day 6). The data in Table 3 indicate that three injections of 0.05 or 0.1 ml almost eliminate splenic suppressor cell activity (Table 3, lines 3 and 4). In an attempt to conserve the anti-I-J sera, in the next experiment, we treated Bl0.A (3R) mice with 0.1 ml anti-I-Jb on the day of birth (Day 0) and tested the mice 3 days later (Day 3). The data in Table 4 indicate that the injection of either conventionally prepared (Table 4, line 3) or monoclonal Table 4, line 4) anti-I-Jb antibody into newly born mice results in decreased splenic suppressor cell activity. A similar type of experiment was performed by injecting monoclonal anti-I-Jb antibody into neonatal C57BL/6 mice. The splenic suppressor cell activity, which is present on Day 4 in the untreated mice (Table 5, line 2) is reduced significantly by neonatal injection of anti-I-Jb antibody (Table 5, line 3). The data in Tables 3-5 therefore suggest that this methodology can be used to test both the early and longer range effects of specific anti-I-J treatment on the relationship between suppressor cell activity and mixed-lymphocyte reactivity. In the next experiment, we injected BIO.A (3R) mice on Day 0 with 0.1 ml of monoclonal anti-I-Jb antibody and tested the mice on Day 4. The data indicate that suppressor cell activity, as before, is decreased (Table 6, line 6). We also see that the percentage responsiveness of the mixed-lymphocyte reactivity, after injection with anti-I-Jb antibody, rises slightly but significantly (Table 6, line 3). For this reason we deemed it worthwhile to test the longer term effects of neonatal in vivo anti-I-J treatment and determined whether the emergence of mixed-lymphocyte reactivity (and immunocompetence in general) is hastened by the antiserum treatment. In the next experiment C57BL/6 mice were injected ip with 0.1 ml of monoclonal anti-I-Jb on Day 0. The mice were sacrificed at intervals and tested for in vitro
1
a-1-J’ + C’ a-1-J’ + C’
Neonatal Neonatal
Adult Neonatal Neonatal Adult Adult
1068 1548 1400 1068 1068
+ 123 f 266 -+ 438 + 123 + 123
AAm + f f k +
A&n 56,224 9,298 4,386 19,720 46,272
’ Spleen cells from 4-day-old Bl0.A (5R) mice treated with antiserum and complement. b P values are compared to untreated neonatal spleen cells.
Treatment of responding or added spleen
Added spleen (5 x 105)
Responding spleen in MLC (5 X 10J) 2268 945 160 1877 2455
[‘H]TdR incorporation in MLC (cpm + SE)
52.6 6.0 3.1 17.3 40.5
SI 100 14 5 -
Percentage response
rO.05
Pb
84 18
Percentage suppression
Treatment of Bl0.A (5R) Neonatal Spleen Cells with cy-I-JkAntiserum and Complement (C’) Decreases Splenic Suppressor Cell Activity but Does Not Restore Mixed-Lymphocyte Reactivity’
TABLE
=O.Ol
Pb
Y
!I
2 g
s
5
3 0
%
z
C’ a-I-Jb + C’
C’ a-I-Jb + C’
Treatment of responding or added spleen
4,409 + 405 4,409 f 405 4,409 + 405
5,253 + 260 13,469 + 1702 10,496 f 734
AAm
AfAn
44,314 + 2994 1,023 + 148 31,580 k 2199
45,833 k 2810 14,522 + 389 15,618 + 739
’ Spleen cells from 4-day old Bl0.A (3R) mice treated with antiserum and complement. b P values are compared to untreated neonatal spleen cells.
Adult Neonatal Neonatal
-
Adult Neonatal Neonatal
Adult Adult Adult
Added spleen (5 x 105)
Responding spleen in MLC (5 X 10’)
[‘H]TdR incorporation in MLC (cpm 2 SE)
10.1 0.2 7.2
8.7 1.1 1.5
SI
-
100 3 13
Percentage response
>0.05
Pb
0 100 32
-
Percentage suppression
Treatment of Bl0.A (3R) Neonatal Spleen Cells with a-1-J” Antiserum and Complement (C’) Decreases Splenic Suppressor Cell Activity but Does Not Restore Mixed-Lymphocyte Reactivity’
TABLE 2
Pb
2 r; % $
g
% 2 E
I-J AND NEONATAL
273
SUPPRESSION
TABLE 3 Injection of BI0.A (5R) Neonatal Mice with (Y-I-J’ Antiserum Decreases Splenic Suppressor Cell Activity” [‘H]TdR incorporation in MLC (cpm + SE) Volume of serum injected (ml)
Added spleen cells (2.5 x 105)
-
Adult Neonatal Neonatal Neonatal
0.05 0.1
AAm 6091 6091 6091 6091
+ + k k
SI
Percentage suppression
Pb
5.6 0.01 5.3 5.2
0 loo 7 9
‘f%
1314 1314 1314 1314
34,271 68 32,221 31,785
+ 1601 + 27 + 2270 + 1238
’ Neonatal mice injected with antiserum on Days 1, 2, and 3 and tested on Day 6. ‘P values compared to neonatal spleen cells from untreated donors (line 2).
responsiveness to DBA/2 alloantigens. Spleen cells from 3-month-old C57BL/6 mice were used as control responder cells and their MLC response set at 100%. The data in Fig. 1 indicate that injection of anti-I-Jb serum into neonatal mice does result in a significant increase of MLC reactivity on the part of the spleen cells at all stages tested. At 45 days of age the spleen cells from the neonatally injected mice have reached 88% of the control level while the noninjected littermates are still at 53% of the control level. Similar results were obtained when neonatal BIO.A (3R) mice were injected on the day of birth with 0. I ml of monoclonal anti-I-Jb antibody and tested at intervals thereafter (Fig. 2). As reported in an earlier publication (2), the mixed-lymphocyte reactivity in nontreated (control) BIO.A (3R) mice develops slower than in C57BL/ 6 mice. Compared to the control mice, mixed-lymphocyte reactivity in the injected (experimental) B 1O.A (3R) mice develops significantly faster. In the next experiment we injected Bl0.A (5R) neonates with 0.1 ml of anti-I-Jk TABLE 4 Injection of BI0.A (3R) Neonatal Mice with cy-I-JbAntibody Decreases Splenic Suppressor Cell Activity’ [‘H]TdR incorporation in MLC (cpm + SE) Treatment of donors
Added spleen cells (5 x 105)
-
Adult Neonatal Neonatal Neonatal
Antiserum’ Antibodyd
‘wn 6124 6124 6124 6124
+ f * f
Ahi 329 329 329 329
62,952 24,317 41,027 44,252
+ f k +
213 1034 872 622
SI
Percentage suppression
Pb
10.3 2.7 4.6 4.9
0 68 39 33
’ Neonatal mice injected ip with 0.1 ml of antiserum on Day 0 and tested on Day 3. b P values are compared to neonatal spleen cells from untreated donors (line 2). ’ cy-I-Jbprepared in the laboratory of Dr. Bertie F. Argyris by injecting Bl0.A (5R) mice with Bl0.A (3R) spleen cells. d cu-I-Jbmonoclonal (WF 9.20.8) antibody prepared in the laboratory of Dr. Carl Waltenbaugh.
274
ARGYRIS
AND WALTENBAUGH TABLE 5
Iniection of C57BL/6 Neonatal Mice with my-I-JbAntibody Decreases Splenic Suppressor Cell Activity [3H]TdR incorporation in MLC (cpm f SE) Treatment of donors”
Added spleen cells (XIOS)
fwn
A&m
SI
Percentage suppression
Pb
-
Adult Neonatal Neonatal
3641 t- 482 3641 3641 ff 482 482
21,618 f 725 1,232 + 91 15,700 + 1066
5.9 0.3 4.3
0 100 33
to.0 1
Anti-IJb
0 Neonatal mice injected ip with 0.1 ml of monoclonal by-I-Jbantibody (WF 9.20.8) on Day 0 and tested on Day 4. b P values are compared to neonatal spleen cells from untreated donors (line 2).
antiserum. Again this causes an increased mixed-lymphocyte reactivity (Fig. 3). In the BIO.A (5R) mice, the experimental and control curves diverge slightly later (after 10 days of age) than in the C57BL/6 and BIO.A (3R) mice which already show a signifkant difference 5 days alter injection (Figs. 1 and 2). This difference may be due to the fact that the C57BL/6 and B 1O.A (3R) mice were injected with monoclonal anti-I-Jb whereas the BIO.A (5R) mice were treated with conventionally prepared anti-I-Jk serum. DISCUSSION Our aim in this paper was to determine whether a decrease in neonatal suppressor cell activity is accompanied by an increase in immunocompetence. Neonatal suppressor cell activity was assayed by adding the cells, treated with mitomycin C, to mixedlymphocyte cultures and measuring the decrease in mixed-lymphocyte reactivity. Previously we have shown that the neonatal splenic suppressor cell activity is mitomycin C resistant (1, 3). We have also shown that suppressor cell activity is decreased when the neonatal spleen cells are preincubated with anti-I-J serum of the appropriate phenotype (3), indicating that the neonatal suppressor cells carry I-J antigen on their surface. To assay for immunocompetence, we measured the mixed-lymphocyte reactivity of the neonatal spleen cells. The data indicate that in vitro treatment of neonatal spleen cells with specific antiI-J sera and complement does decrease suppressor cell activity but does not cause an immediate increase in mixed-lymphocyte reactivity of the neonatal spleen cells. But there is a delayed increase in mixed-lymphocyte reactivity as was demonstrated by removing and testing spleen cells at intervals after neonatal injection of anti-I-J serum. Thus in vivo treatment of neonates with anti-I-J antibody hastens the development of mixed-lymphocyte reactivity. Since mixed-lymphocyte reactivity is an in vitro example of cell-mediated immunity (1 l), we can broaden our conclusion and suggest that removal of neonatal suppressor cells hastens the development of cellmediated immunity and possibly the immune response in general. Our results on cell-mediated immunity fit those of Murgita et al. (4) who showed that injection of anti-I-J sera into neonatal mice leads to an increase in the number of splenic plaqueforming cells 20 days later.
Adult Neonatal Neonatal
Adult Neonatal Neonatal Adult Adult Adult
a-I-Jb &I-Jb
Treatment of donors of responding or added spleen 8610 5161 5231 6583 6583 6583
+ + + + r +
AAm 931 136 530 475 415 475
59,443 4,661 8,306 39,645 7,699 17,945
+ 2051 +- 343 +- 244 -L 2619 A 433 k 464
A&n 6.9 0.9 1.6 6.0 1.2 2.1
SI
’ Neonatal mice injected on Day 0 with 0.1 ml of monoclonal antibody (WF 9.20.8) and tested on Day 4. b P values are compared to untreated neonatal mice.
Added spleen (2.5 x 105)
Responding spleen in MLC (5 X 10’)
[3H]TdR incorporation in MLC (cpm k SE)
-
100 0 6 -
Percentage response
co.0 I
Pb
Injection of c+I-Jb Antiserum into Neonatal Bl0.A (3R) Mice Decreases Suppressor Cell Activity and Increases the Mixed-Lymphocyte
TABLE 6
0 91 64
-
Percentage suppression
Pb
KO.01
Reactivity Significantly”
g z
z > 3
276
ARGYRIS
AND WALTENBAUGH
so.
SO.
70.
so.
50.
40. w z 230. P 0 d20. I 8
I
.--** 4 5 10 AGE
20
15 (DAYS)
25
30
35
FIG. 1. C57BL/6 neonatal mice injected with 0.1 ml monoclonal anti-I-Jb (WF 9.20.8) and tested at intervals. X - X, injected; 0 - - - 0, control.
The data therefore suggest that removal of neonatal suppressor cell activity results in a gradual, but definite increase in immunocompetence. A slow recovery of immunoreactivity after removal of suppressor cells has also been reported by Green and Gershon (12). These investigators, studying contact sensitivity to picryl chloride in adult mice, cultured T-helper and T-suppressor cells in separate compartments of Marbrook chambers. They found that removal of the suppressor cells did not result in an immediate recovery of helper cell activity.
w40. z
a
f30. (E 2 i20.
/
0a@ 10.
,.-
/ -A
--
_---
-0
.I‘ 3’
L.
,e-.
5
/@I 10
AGE
15
20
25
30
35
(DAYS)
2. B 10.A (3R) neonatal mice injected with 0.1 ml monoclonal anti-I-Jb (WF 9.20.8) and tested at intervals. X - X, injected; 0 - - - 0, control. FIG.
I-J AND NEONATAL
277
SUPPRESSION
0-r20. I ap 10.
5
10
15
20 AGE
25 (DAYS
30
35
40
45
4 50
1
FIG. 3. Bl0.A (5R) neonatal mice injected with 0.1 ml anti-I-J’ serum and tested at intervals. X - X, injected, 0 - - - 0, control.
The mechanism of immunological immaturity in the neonatal mouse is not known. Several investigators (13-l 6) have shown that functional T and B lymphocytes in the mouse are present at birth. These immunocompetent cells may be prevented from functioning by the suppressor cells. A gradual decrease in suppressor cell activity may be responsible for a gradual increase in immunocompotence after birth (2). We have presented evidence that administration of adult macrophages to neonatal mice decreases suppressor cell prematurely (5, 17). We have suggested that the number and/or maturity of macrophages in the neonate may control, at least in part, the activity of suppressor cells and in turn the development of immunocompetence. The present data with anti-I-J antibody fit our hypothesis. The anti-I-J antibodies used in our studies were both monoclonal and conventionally produced antisera. The latter were produced in congenic B 1O.A (3R) (I-Jb) and B 1O.A (5R) (1-J’) mice. We find that 7-8 weekly ip injections of 50 X lo6 spleen ceils are required for raising a respectable antibody titer. We find that the removal of suppressor cell activity from neonatal spleen cells after treatment with anti-I-J and complement is a relatively simple and reliable assay for anti-I-J sera. In summary, the data in this paper show that (1) neonatal spleen cells can be used as a reliable assay for anti-I-J sera and (2) I-J-positive suppressor cells in the neonatal mouse play an important role in controlling the development of immunocompetence. ACKNOWLEDGMENTS The competent laboratory assistance of Michelle DeStefano, Laura Love& and Sandra Bogdon is gratefully acknowledged.
REFERENCES 1. 2. 3. 4.
A&s, Argyris, Argyris, Murgita, 957,
B. F., Cell. Immunol. 48, 398, 1979. B. F., Transplantation 31, 334, 1981. B. F., Cell. Immunol. 66, 352, 1982. R. A., Hooper, D. C., Stegagno, M., Delovitch, T. L., and Wigzell, H., Eur. J. Immunol. 11, 1981.
ARGYRJS AND WALTENBAUGH
278 5. 6. 7. 8. 9. 10. Il.
Argyris, B. F., Cell. Immunol. 74, 313, 1982. Rodriguez, G., Andersson, B., Wig&l, H., and Peck, A. B., Eur. J. Immunol. 9,131, 1979. Piguet, P. F., Irle, C., and Vassalli, P., Eur. J. Immunol. 11, 56, 1981. Snyder, D. S., Lu, C. Y., and Unanue, E. R., J. Immunol. 128, 1458, 1982. Boyle, W., Transplantation 6, 761, 1968. Waltenbaugh, C., J. Exp. Med. 154, 1570, 1981. Bach, F. H., Bock, H., Graupner, K., Day, E., and Klostermann, H., Proc. Natl. Acad. Sci. USA 62,
12. 13.
Green, D. R., and Gershon, R. K., Ann. N. Y. Acad. Sci. 392, 3 18, 1982. Spear, P. G., Wang, A. L., Rutishauser, V., and Edelman, G. M., J. Exp. Med. 138, 557, 1973. Press, J. L., and Klinman, N. R., Eur. J. Immunol. 4, 155, 1974. Pilarski, L. M., J. Exp. Med. 146, 887, 1977. Haines, K. A., and Siskind, G. W., J. Immunol. 124, 1878, 1980. Argyris, B. F., Transplantation, in press, 1983.
377, 1969.
14. 15. 16. 17.
IMMUNOLOGY
80, 267-278
(1983)
Role of I-J in Neonatal BERTIE F. ARGYRIS~ANDCARL
Suppression’ WALTENBAUGH
Department of Microbiology, Upstate Medical Center, Syracuse, New York 13210, and Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611 Received March 4, 1983; accepted May 17, 1983 Spleen cells from neonatal mice belonging to strains with the I-J-b or I-J-’ genotype, were treated with anti-I-Jb or anti-I-J’ antibody and complement. This reduces their suppressor cell activity as demonstrated by a decrease in the ability to suppress the mixed-lymphocyte reactivity of adult spleen cells. Injection of anti-I-J antibody into neonatal mice also reduces splenic suppressor cell activity prematurely. The removal of suppressor cells from neonatal spleen does not result in an immediate increase in mixed-lymphocyte reactivity (cell-mediated immunity) but does hasten the development of mixed-lymphocyte reactivity in the young mice. The results are discussed in light of the hypothesis that suppressor cells inhibit the function of immunocompetent cells in the neonatal mouse and control the development of immunocompetence.
INTRODUCTION Newborn mice are immunologically immature. The immunological immaturity has, at least in part, been contributed to high suppressor cell activity (l-4) and low macrophage function (5). The suppressor cell in the spleen of newborn mice has been shown to be an I-J+, Lyt-l+, Lyt-2- T cell (3, 4) but several investigators claim that neonatal splenic non-T cells also have suppressor cell activity (6-8). We have shown that the splenic suppressor cell activity in postnatal mice is inversely correlated with the degree of immunological maturity (2). We have presented evidence which suggests strongly that a gradual increase in the number and/or function of macrophages after birth results in a gradual decrease in suppressor cell activity (2, 5). We have postulated that this gradual decrease in suppressor cell activity allows for immunocompetence to emerge. In this paper we are testing this hypothesis. We have treated neonatal spleen cells with anti-I-J antibody and determined whether the resulting decrease in suppressor cell activity is accompanied by an increase in immunocompetence, as measured by mixed-lymphocyte reactivity. We have also injected neonatal mice with anti-I-J antibody to test whether this decreases suppressor cell activity and subsequently leads to earlier development of immunocompetence. ’ This work was supported by USPHS Research Grant CA 15462 from the National Cancer Institute and Grant AI 18072 from National Institutes of Health. ’ To whom requests for reprints should be addressed. 267 0008-8749/83
$3.00
Copyngbt 0 1983 by Academic Press, Inc. All rights of reproduction m any form reserved.
268
ARGYRIS
AND
MATERIALS
WALTENBAUGH
AND
METHODS
Mice. Adult C57BL/6, DBA/2, and adult B6D2F1 mice were purchased from Jackson Laboratories, Bar Harbor, Maine, as 7-week-old animals, and used at 12 weeks of age. Bl0.A (3R) and BIO.A (5R) were obtained as breeding pairs from Dr. Jack Stimpfling and Dr. Phillippa Marrack, respectively. They were bred by brother and sister matings in our laboratory. Neonatal mice were obtained from our own colony. BDF, mice are the Fi offspring from C57BL/6 female and DBA/2 male mice. Cell suspensions. Single-cell suspensions were prepared by pressing spleens through size 50 stainless-steel mesh into phosphate-buffered saline. Red blood cells, from adult spleens only, were lysed by hypotonic shock (9). Cells were counted in 0.5% acetic acid and suspended in RPM1 1640 medium (Gibco), supplemented with 25 mM Hepes, 10 pg/lOO ml streptomycin, 10,000 U/ 100 ml penicillin, and 5 mg/lOO ml gentamicin. Mixed-lymphocyte culture (MLC). Mixed-lymphocyte cultures were carried out as described previously (3) with 3 X lo5 spleen cells responding to 3 X 10’ DBA/2 stimulating spleen cells in a total volume of 0.2 ml tissue culture medium (RPMI) with 10% of fetal calf serum. Up to 100 X lo6 stimulating cells were pretreated with 50 kg mitomycin C (kindly donated to us by Dr. William Bradner, Bristol Laboratories, Syracuse, N.Y.), in 2 ml of phosphate-buffered saline, for 20 min in a 37°C shaking water bath. The mitomycin-treated cells were washed three times before use. This dose of mitomycin is adequate to prevent incorporation of [3H]TdR into lymphocytes. Cultures were labeled with 1 PCi of [3H]TdR (Amersham) after 72 hr and harvested with an automatic cell harvester (0. Hiller) after 96 hr. Uptake of [3H]TdR by the proliferating cells was determined in a Packard scintillation counter. Triplicate cultures of syngeneic (AA,) and allogeneic (AB,) cells were carried out. The stimulation index (SI) represents the ratio of average counts in AB, and AA,,, cultures. The percentage response of the adult responding cells (control) was arbitrarily set at 100%. The mixed-lymphocyte reactivity of neonatal responding spleen cells was compared to that of syngeneic adult responding spleen cells (donors 3-4 months of age) and expressed as percentage responsiveness by calculating the ratio of the differences in counts per minute between AB, and AA,,, cultures, e.g., % response =
AB, - AA,,, (neonatal) x loo AB, - AA,,, (adult) ’
where adult denotes the use of adult responding cells and neonatal denotes the use of neonatal responding cells in MLC. Measurement of suppressor cell activity. The suppressor cell activity of neonatal spleen cells was measured as described previously (3). In short, 5 and 2.5 X 10’ mitomycin-treated neonatal spleen cells were added to MLC assay cultures of 3 X 10’ adult syngeneic spleen cells responding to 3 X 10’ DBA/2 mitomycin-treated stimulating spleen cells. Previously we have shown that the suppressor cell activity of neonatal spleen cells is mitomycin resistant (1, 3). The control cultures received an equal number of mitomycin-treated spleen cells from adult mice syngeneic to the responding spleen cells. Mitomycin treatment of the added cells was carried out as described above for the stimulating spleen cells in MLC. The percentage suppression was calculated as follows
I-J AND NEONATAL
% suppression = 100 -
SUPPRESSION
269
AB, - AA, (neonatal) x 100 AB, - AA,,, (adult) I ’
where adult denotes cultures receiving added adult cells and neonatal denotes cultures to which neonatal cells were added. Production of anti-Z-J sera. “Conventional” anti-I-J sera were prepared by weekly ip injections of Bl0.A (3R) spleen cells into BIO.A (5R) mice for anti-I-Jb and in the reverse direction for anti-I-Jk. Fifty million spleen cells were injected each time. In most cases the mice started producing an effective antiserum after 10-l 1 weeks. Serum was collected by bleeding the mice from the orbital cavity. The sera were tested by measuring loss of suppressor activity of neonatal spleen cells (Bl0.A (3R) for a-I-Jb and BIO.A (5R) for a-I-Jk). Sera were stored in small aliquots at -6O“C. Production of monoclonal anti-Z-P. Monoclonal anti-I-J-b antibodies (WF 9.20.8) have been previously characterized ( 10). The antibodies were used as a culture fluid diluted in RPM1 medium. Treatment with anti-Z-J sera and complement. Thirty to fifty million neonatal spleen cells were incubated with a I: 10 dilution of the appropriate (Y-I-J serum for 1 hr at 4°C. The cells were centrifuged, resuspended in a 1: 10 dilution of rabbit complement (Low-Tox; Cedar Lanes), and reincubated for l/2 hr in a 37°C shaking water bath. Control tubes included neonatal cells treated with RPM1 or RPM1 and complement. The cells were washed three times. Half of the cell suspension was used for the measurement of mixed-lymphocyte reactivity (see above); the other half of the cell suspension was treated with mitomycin (see above) and used for the measurement of suppressor cell activity (see above). Injection of anti-Z-J antibody. Neonatal mice were injected ip with 0.1 ml of antibody via a dorsal approach. The injection site was sealed with a surgical spray to prevent leakage of the injected material. Three or four mice from each litter were injected with the same aliquot and their spleen cells pooled at the time of sacrifice. The neonates were marked by clipping the tail and/or right/left toe nails. The control mice were injected with phosphate-buffered saline only. After injection, the neonates were returned to their mothers and kept as an isolated litter until they were sacrificed. Spleen cells from each group were pooled and made into a single-cell suspension. One half of the neonatal cell suspension was treated with mitomycin (see above) and used for the measurement of suppressor cell activity (see above). The other half of the cell suspension was used to measure mixed-lymphocyte reactivity, using mitomycintreated DBA/2 spleen cells as stimulating spleen cells. Only the adult and not the neonatal spleen cells were subjected to hypotonic shock, for removal of red blood cells. Presentation of data. The tables contain representative data from one experiment. All experiments were repeated three to four times. Statistical analysis was carried out with the small-sample method of Fisher. RESULTS Previously (3) we have demonstrated that treatment of neonatal spleen cells with specific anti-I-J sera results in decreased suppressor cell activity. At that time we also showed that the effect was specific because BIO.A (3R) neonatal spleen cells (I-Jb)
270
ARGYRIS
AND
WALTENBAUGH
treated with anti-I-Jb serum and complement lost suppressor cell activity whereas treatment with anti-I-Jk had no effect (3). In the following experiments we determine whether this decrease in suppressor cell activity results in an increased mixed-lymphocyte reactivity. The data in Table 1 confirm our previous results (3) and indicate that the high suppressor celI activity (Table 1, line 4) in neonatal B 1O.A (5R) mice is decreased considerably after treating the neonatal spleen cells with anti-I-Jk serum and complement (Table 1, line 5). However, when we use these neonatal spleen cells as responder cells in mixed-lymphocyte cultures we do not see a significant change in percentage responsiveness (Table 1, lines 2 and 3). Similar data are presented in Table 2 where spleen cells from neonatal B1O.A (3R) mice were treated with specific anti-I-Jb serum and complement. The results indicate that suppressor cell activity is lost (Table 2, lines 5 and 6), but percentage responsiveness of mixed-lymphocyte reactivity does not rise significantly (Table 2, lines 2 and 3). The data in Tables 1 and 2 suggest that (a) suppressor cell activity and mixedlymphocyte reactivity are unrelated or (b) a time interval is required for the immunocompetent cells to escape from the suppressive influence of the suppressor cells. To distinguish between these two possibilities, we changed our methodology and injected the neonatal mice with anti-I-J antisera in order to follow the events taking place in vivo. In the experiment depicted in Table 3, we injected BIO.A (5R) mice ip on the frrst, second, and third days alter birth (days 1,2, and 3) with 0.1 ml of anti-I-Jk antiserum and tested the splenic suppressor cell activity 3 days later (Day 6). The data in Table 3 indicate that three injections of 0.05 or 0.1 ml almost eliminate splenic suppressor cell activity (Table 3, lines 3 and 4). In an attempt to conserve the anti-I-J sera, in the next experiment, we treated Bl0.A (3R) mice with 0.1 ml anti-I-Jb on the day of birth (Day 0) and tested the mice 3 days later (Day 3). The data in Table 4 indicate that the injection of either conventionally prepared (Table 4, line 3) or monoclonal Table 4, line 4) anti-I-Jb antibody into newly born mice results in decreased splenic suppressor cell activity. A similar type of experiment was performed by injecting monoclonal anti-I-Jb antibody into neonatal C57BL/6 mice. The splenic suppressor cell activity, which is present on Day 4 in the untreated mice (Table 5, line 2) is reduced significantly by neonatal injection of anti-I-Jb antibody (Table 5, line 3). The data in Tables 3-5 therefore suggest that this methodology can be used to test both the early and longer range effects of specific anti-I-J treatment on the relationship between suppressor cell activity and mixed-lymphocyte reactivity. In the next experiment, we injected BIO.A (3R) mice on Day 0 with 0.1 ml of monoclonal anti-I-Jb antibody and tested the mice on Day 4. The data indicate that suppressor cell activity, as before, is decreased (Table 6, line 6). We also see that the percentage responsiveness of the mixed-lymphocyte reactivity, after injection with anti-I-Jb antibody, rises slightly but significantly (Table 6, line 3). For this reason we deemed it worthwhile to test the longer term effects of neonatal in vivo anti-I-J treatment and determined whether the emergence of mixed-lymphocyte reactivity (and immunocompetence in general) is hastened by the antiserum treatment. In the next experiment C57BL/6 mice were injected ip with 0.1 ml of monoclonal anti-I-Jb on Day 0. The mice were sacrificed at intervals and tested for in vitro
1
a-1-J’ + C’ a-1-J’ + C’
Neonatal Neonatal
Adult Neonatal Neonatal Adult Adult
1068 1548 1400 1068 1068
+ 123 f 266 -+ 438 + 123 + 123
AAm + f f k +
A&n 56,224 9,298 4,386 19,720 46,272
’ Spleen cells from 4-day-old Bl0.A (5R) mice treated with antiserum and complement. b P values are compared to untreated neonatal spleen cells.
Treatment of responding or added spleen
Added spleen (5 x 105)
Responding spleen in MLC (5 X 10J) 2268 945 160 1877 2455
[‘H]TdR incorporation in MLC (cpm + SE)
52.6 6.0 3.1 17.3 40.5
SI 100 14 5 -
Percentage response
rO.05
Pb
84 18
Percentage suppression
Treatment of Bl0.A (5R) Neonatal Spleen Cells with cy-I-JkAntiserum and Complement (C’) Decreases Splenic Suppressor Cell Activity but Does Not Restore Mixed-Lymphocyte Reactivity’
TABLE
=O.Ol
Pb
Y
!I
2 g
s
5
3 0
%
z
C’ a-I-Jb + C’
C’ a-I-Jb + C’
Treatment of responding or added spleen
4,409 + 405 4,409 f 405 4,409 + 405
5,253 + 260 13,469 + 1702 10,496 f 734
AAm
AfAn
44,314 + 2994 1,023 + 148 31,580 k 2199
45,833 k 2810 14,522 + 389 15,618 + 739
’ Spleen cells from 4-day old Bl0.A (3R) mice treated with antiserum and complement. b P values are compared to untreated neonatal spleen cells.
Adult Neonatal Neonatal
-
Adult Neonatal Neonatal
Adult Adult Adult
Added spleen (5 x 105)
Responding spleen in MLC (5 X 10’)
[‘H]TdR incorporation in MLC (cpm 2 SE)
10.1 0.2 7.2
8.7 1.1 1.5
SI
-
100 3 13
Percentage response
>0.05
Pb
0 100 32
-
Percentage suppression
Treatment of Bl0.A (3R) Neonatal Spleen Cells with a-1-J” Antiserum and Complement (C’) Decreases Splenic Suppressor Cell Activity but Does Not Restore Mixed-Lymphocyte Reactivity’
TABLE 2
Pb
2 r; % $
g
% 2 E
I-J AND NEONATAL
273
SUPPRESSION
TABLE 3 Injection of BI0.A (5R) Neonatal Mice with (Y-I-J’ Antiserum Decreases Splenic Suppressor Cell Activity” [‘H]TdR incorporation in MLC (cpm + SE) Volume of serum injected (ml)
Added spleen cells (2.5 x 105)
-
Adult Neonatal Neonatal Neonatal
0.05 0.1
AAm 6091 6091 6091 6091
+ + k k
SI
Percentage suppression
Pb
5.6 0.01 5.3 5.2
0 loo 7 9
‘f%
1314 1314 1314 1314
34,271 68 32,221 31,785
+ 1601 + 27 + 2270 + 1238
’ Neonatal mice injected with antiserum on Days 1, 2, and 3 and tested on Day 6. ‘P values compared to neonatal spleen cells from untreated donors (line 2).
responsiveness to DBA/2 alloantigens. Spleen cells from 3-month-old C57BL/6 mice were used as control responder cells and their MLC response set at 100%. The data in Fig. 1 indicate that injection of anti-I-Jb serum into neonatal mice does result in a significant increase of MLC reactivity on the part of the spleen cells at all stages tested. At 45 days of age the spleen cells from the neonatally injected mice have reached 88% of the control level while the noninjected littermates are still at 53% of the control level. Similar results were obtained when neonatal BIO.A (3R) mice were injected on the day of birth with 0. I ml of monoclonal anti-I-Jb antibody and tested at intervals thereafter (Fig. 2). As reported in an earlier publication (2), the mixed-lymphocyte reactivity in nontreated (control) BIO.A (3R) mice develops slower than in C57BL/ 6 mice. Compared to the control mice, mixed-lymphocyte reactivity in the injected (experimental) B 1O.A (3R) mice develops significantly faster. In the next experiment we injected Bl0.A (5R) neonates with 0.1 ml of anti-I-Jk TABLE 4 Injection of BI0.A (3R) Neonatal Mice with cy-I-JbAntibody Decreases Splenic Suppressor Cell Activity’ [‘H]TdR incorporation in MLC (cpm + SE) Treatment of donors
Added spleen cells (5 x 105)
-
Adult Neonatal Neonatal Neonatal
Antiserum’ Antibodyd
‘wn 6124 6124 6124 6124
+ f * f
Ahi 329 329 329 329
62,952 24,317 41,027 44,252
+ f k +
213 1034 872 622
SI
Percentage suppression
Pb
10.3 2.7 4.6 4.9
0 68 39 33
’ Neonatal mice injected ip with 0.1 ml of antiserum on Day 0 and tested on Day 3. b P values are compared to neonatal spleen cells from untreated donors (line 2). ’ cy-I-Jbprepared in the laboratory of Dr. Bertie F. Argyris by injecting Bl0.A (5R) mice with Bl0.A (3R) spleen cells. d cu-I-Jbmonoclonal (WF 9.20.8) antibody prepared in the laboratory of Dr. Carl Waltenbaugh.
274
ARGYRIS
AND WALTENBAUGH TABLE 5
Iniection of C57BL/6 Neonatal Mice with my-I-JbAntibody Decreases Splenic Suppressor Cell Activity [3H]TdR incorporation in MLC (cpm f SE) Treatment of donors”
Added spleen cells (XIOS)
fwn
A&m
SI
Percentage suppression
Pb
-
Adult Neonatal Neonatal
3641 t- 482 3641 3641 ff 482 482
21,618 f 725 1,232 + 91 15,700 + 1066
5.9 0.3 4.3
0 100 33
to.0 1
Anti-IJb
0 Neonatal mice injected ip with 0.1 ml of monoclonal by-I-Jbantibody (WF 9.20.8) on Day 0 and tested on Day 4. b P values are compared to neonatal spleen cells from untreated donors (line 2).
antiserum. Again this causes an increased mixed-lymphocyte reactivity (Fig. 3). In the BIO.A (5R) mice, the experimental and control curves diverge slightly later (after 10 days of age) than in the C57BL/6 and BIO.A (3R) mice which already show a signifkant difference 5 days alter injection (Figs. 1 and 2). This difference may be due to the fact that the C57BL/6 and B 1O.A (3R) mice were injected with monoclonal anti-I-Jb whereas the BIO.A (5R) mice were treated with conventionally prepared anti-I-Jk serum. DISCUSSION Our aim in this paper was to determine whether a decrease in neonatal suppressor cell activity is accompanied by an increase in immunocompetence. Neonatal suppressor cell activity was assayed by adding the cells, treated with mitomycin C, to mixedlymphocyte cultures and measuring the decrease in mixed-lymphocyte reactivity. Previously we have shown that the neonatal splenic suppressor cell activity is mitomycin C resistant (1, 3). We have also shown that suppressor cell activity is decreased when the neonatal spleen cells are preincubated with anti-I-J serum of the appropriate phenotype (3), indicating that the neonatal suppressor cells carry I-J antigen on their surface. To assay for immunocompetence, we measured the mixed-lymphocyte reactivity of the neonatal spleen cells. The data indicate that in vitro treatment of neonatal spleen cells with specific antiI-J sera and complement does decrease suppressor cell activity but does not cause an immediate increase in mixed-lymphocyte reactivity of the neonatal spleen cells. But there is a delayed increase in mixed-lymphocyte reactivity as was demonstrated by removing and testing spleen cells at intervals after neonatal injection of anti-I-J serum. Thus in vivo treatment of neonates with anti-I-J antibody hastens the development of mixed-lymphocyte reactivity. Since mixed-lymphocyte reactivity is an in vitro example of cell-mediated immunity (1 l), we can broaden our conclusion and suggest that removal of neonatal suppressor cells hastens the development of cellmediated immunity and possibly the immune response in general. Our results on cell-mediated immunity fit those of Murgita et al. (4) who showed that injection of anti-I-J sera into neonatal mice leads to an increase in the number of splenic plaqueforming cells 20 days later.
Adult Neonatal Neonatal
Adult Neonatal Neonatal Adult Adult Adult
a-I-Jb &I-Jb
Treatment of donors of responding or added spleen 8610 5161 5231 6583 6583 6583
+ + + + r +
AAm 931 136 530 475 415 475
59,443 4,661 8,306 39,645 7,699 17,945
+ 2051 +- 343 +- 244 -L 2619 A 433 k 464
A&n 6.9 0.9 1.6 6.0 1.2 2.1
SI
’ Neonatal mice injected on Day 0 with 0.1 ml of monoclonal antibody (WF 9.20.8) and tested on Day 4. b P values are compared to untreated neonatal mice.
Added spleen (2.5 x 105)
Responding spleen in MLC (5 X 10’)
[3H]TdR incorporation in MLC (cpm k SE)
-
100 0 6 -
Percentage response
co.0 I
Pb
Injection of c+I-Jb Antiserum into Neonatal Bl0.A (3R) Mice Decreases Suppressor Cell Activity and Increases the Mixed-Lymphocyte
TABLE 6
0 91 64
-
Percentage suppression
Pb
KO.01
Reactivity Significantly”
g z
z > 3
276
ARGYRIS
AND WALTENBAUGH
so.
SO.
70.
so.
50.
40. w z 230. P 0 d20. I 8
I
.--** 4 5 10 AGE
20
15 (DAYS)
25
30
35
FIG. 1. C57BL/6 neonatal mice injected with 0.1 ml monoclonal anti-I-Jb (WF 9.20.8) and tested at intervals. X - X, injected; 0 - - - 0, control.
The data therefore suggest that removal of neonatal suppressor cell activity results in a gradual, but definite increase in immunocompetence. A slow recovery of immunoreactivity after removal of suppressor cells has also been reported by Green and Gershon (12). These investigators, studying contact sensitivity to picryl chloride in adult mice, cultured T-helper and T-suppressor cells in separate compartments of Marbrook chambers. They found that removal of the suppressor cells did not result in an immediate recovery of helper cell activity.
w40. z
a
f30. (E 2 i20.
/
0a@ 10.
,.-
/ -A
--
_---
-0
.I‘ 3’
L.
,e-.
5
/@I 10
AGE
15
20
25
30
35
(DAYS)
2. B 10.A (3R) neonatal mice injected with 0.1 ml monoclonal anti-I-Jb (WF 9.20.8) and tested at intervals. X - X, injected; 0 - - - 0, control. FIG.
I-J AND NEONATAL
277
SUPPRESSION
0-r20. I ap 10.
5
10
15
20 AGE
25 (DAYS
30
35
40
45
4 50
1
FIG. 3. Bl0.A (5R) neonatal mice injected with 0.1 ml anti-I-J’ serum and tested at intervals. X - X, injected, 0 - - - 0, control.
The mechanism of immunological immaturity in the neonatal mouse is not known. Several investigators (13-l 6) have shown that functional T and B lymphocytes in the mouse are present at birth. These immunocompetent cells may be prevented from functioning by the suppressor cells. A gradual decrease in suppressor cell activity may be responsible for a gradual increase in immunocompotence after birth (2). We have presented evidence that administration of adult macrophages to neonatal mice decreases suppressor cell prematurely (5, 17). We have suggested that the number and/or maturity of macrophages in the neonate may control, at least in part, the activity of suppressor cells and in turn the development of immunocompetence. The present data with anti-I-J antibody fit our hypothesis. The anti-I-J antibodies used in our studies were both monoclonal and conventionally produced antisera. The latter were produced in congenic B 1O.A (3R) (I-Jb) and B 1O.A (5R) (1-J’) mice. We find that 7-8 weekly ip injections of 50 X lo6 spleen ceils are required for raising a respectable antibody titer. We find that the removal of suppressor cell activity from neonatal spleen cells after treatment with anti-I-J and complement is a relatively simple and reliable assay for anti-I-J sera. In summary, the data in this paper show that (1) neonatal spleen cells can be used as a reliable assay for anti-I-J sera and (2) I-J-positive suppressor cells in the neonatal mouse play an important role in controlling the development of immunocompetence. ACKNOWLEDGMENTS The competent laboratory assistance of Michelle DeStefano, Laura Love& and Sandra Bogdon is gratefully acknowledged.
REFERENCES 1. 2. 3. 4.
A&s, Argyris, Argyris, Murgita, 957,
B. F., Cell. Immunol. 48, 398, 1979. B. F., Transplantation 31, 334, 1981. B. F., Cell. Immunol. 66, 352, 1982. R. A., Hooper, D. C., Stegagno, M., Delovitch, T. L., and Wigzell, H., Eur. J. Immunol. 11, 1981.
ARGYRJS AND WALTENBAUGH
278 5. 6. 7. 8. 9. 10. Il.
Argyris, B. F., Cell. Immunol. 74, 313, 1982. Rodriguez, G., Andersson, B., Wig&l, H., and Peck, A. B., Eur. J. Immunol. 9,131, 1979. Piguet, P. F., Irle, C., and Vassalli, P., Eur. J. Immunol. 11, 56, 1981. Snyder, D. S., Lu, C. Y., and Unanue, E. R., J. Immunol. 128, 1458, 1982. Boyle, W., Transplantation 6, 761, 1968. Waltenbaugh, C., J. Exp. Med. 154, 1570, 1981. Bach, F. H., Bock, H., Graupner, K., Day, E., and Klostermann, H., Proc. Natl. Acad. Sci. USA 62,
12. 13.
Green, D. R., and Gershon, R. K., Ann. N. Y. Acad. Sci. 392, 3 18, 1982. Spear, P. G., Wang, A. L., Rutishauser, V., and Edelman, G. M., J. Exp. Med. 138, 557, 1973. Press, J. L., and Klinman, N. R., Eur. J. Immunol. 4, 155, 1974. Pilarski, L. M., J. Exp. Med. 146, 887, 1977. Haines, K. A., and Siskind, G. W., J. Immunol. 124, 1878, 1980. Argyris, B. F., Transplantation, in press, 1983.
377, 1969.
14. 15. 16. 17.