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B-cell amplification by dibutyryl cyclic AMP in mice
B-Cell Amplification ANELIA Laborator,y
of
by Dibutyryl
D. UZUNOVA
Cyclic AMP in Mice
l, 2 AND EDGAR E. HANNA
Molecular Genetics, National Institute of Child Health National Institutes of Health, Brtlwsda, Mtrr~~la~zti Receizvd
Drcetrlber
and Htrwton 20014
Dazrelopnzettt,
26, 1972
Mke injected with NO-2’-O-dibutyryl-adcnosine 3’,5’ monophosphatc (DBcAMP) showed increased anti-sheep erythrocyte (anti-SE) antibody plaque-forming spleen cell (PFC) responses up to 7-fold greater than control mice. The amount of increase was related to the immunizing dose of SE and to the dose of DBcAMP, and it \vas more pronounced in 19s PFC than in 7s PFC. Mice thymectomized (TX) within 16 hr after birth and injected with SE and DBcAMP at 40 days showed a 2.7-fold greater anti-SE PFC response than TX controls injected with SE alone. DBcAMP restored the PFC response of TX mice to 75% of that seen for sham TX, DBcAMPtreated controls. These data suggest that a T cell-B cell interaction is not stringently requil-ed in mouse anti-SE antibody responses ilc vko, since a T &-like cffcct may he substituted or a minimal response can be enhanced with a soluble amplifier such as dihutyryl cyclic AMP.
INTRODUCTION It has been shown (1, 2) that thymectomized mice show a deficit in their antibndy response to injected sheep erythrocytes (SE). Such findings have led to the conclusion that the SE is a thymus-dependent antigen requiring the complex interaction of at least two cell types, a thymus-influenced, nonantibody secreting lymphoid T cell and a bone marrow-derived precursor of the antibody-secreting cell. An interesting, but complex model involving a physical association of T-cells and imhas been proposed for the munogenic carrier, B-cells and haptenic determinants interaction of these two cell types in response to the more stringent, defined synthetic hal,tens (3). This nlotlel is also assumed 1)~ Inany to be applical)le to tnouse anti-SE responses. However, when it is recalled that the heterophile SE antigencomplex is a superior immune, ven in essentially all vertebrates and is active in some subvertebrates (4), it would appear unnecessary for natural selection of such a complex mechanism as proposed in the model. The data presented here complement the results of some investigations (5) and offers a 1,ossible definition for the active factor reported by others (6), to jointly show
that
cell-free
factors
may
anlplify
the
antibody-secreting
potential
of R cells
in the presence of specific antigen directly. The present data do not question the model’s application in tile mechanism of the stringent synthetic imniunogens (3). 1 Recipient of an NIH Fogerty 2 Present address : Department
International Visiting Fellowship. of Pathophysiology, Higher Medical 507
Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
Institute,
Varna,
Bulgaria.
.3JH
SITOR? (‘OMMX~NTChTIoNS AlATERIALS
AND
METHODS
Mice. c57 BI,/6N males, 6-8 weeks old, averaging 25 g, were fIXJill the NIH central animal facility. C;+Hf/HeN iieunatal mice were obtained from pregnant mothers in our laboratory animal room. Pregnant mothers were supplied as above. This strain was used in the thymcctomy experiment because of their good survival and care by mothers after surgery. Thymectomy (Tx) was performed through a small slit in the neck, by air vacuum, followed by sealing the slit with pernlount (Fisher Scientific). Sham thymectomy included the same l)rocetlure without surking out the thymus tissue. The procedure was carried out under a heat lamp and in the presence of mothers, who were themselves painted with the glue, permount. Experimental mice were injected as indicated 40 days later. Iln?nzlnogen. Sheep blood was obtained in acid citrate solution from the NIH Large Animal ,Facility. Washed sheep erythrocyte (SE) suspensions were prepared by repeated washing in pyrogen-free (7) phosphate-buffered saline (PBS). They were diluted to the desired concentration in Pl3S as noted in the data tables. All injections were made intraperitoneally (ip) . URrAM P, N”-2’-O-dibutyryl-adenosine 3’,5’-cyclic phosphate. sodium. 5H,O, A grade, Lot No. 010162, was obtamecl from Calblochem, Fullerton, CA, and maintained desiccated in the cold. For use it was dissolved in pyrogen-free water and diluted as desired in PBS. Injections were by the ip route. Spleen cell suspensions consisted of whole dispersed spleens pooled from 15-20 mice in each experimental group, shown in the tables to follow. They were made by cutting spleens into small pieces and passing the tissue repeatedly through a 44-pm pore size, stainless-steel mesh cell sieve using ice cold Hanks’ BSS, pH 7.4, as the suspending medium as previously detailed (8). A portion of each cell suspension was cryogenically prepared and frozen ( - 1 “/min) in the I&de electronically controlled, liquid nitrogen biological freezer to facilitate repeat of assays as needed. PFC assays were performed as previously detailed (8), except that mouse IgG SEplaques, recorded as 7S plaques, were developed by use of hyperimmune, &sorbed, rabbit anti-mouse 7S imiiiunoglol~ulin and 109, guinea pig complement in PBS containing optimum Ca’+ and hlg”. Cells were couiited in the Autocytometer I I (Fisher Scientific Co., Pittsburgh, PA), electronic cell counter. RESULTS
AND
DISCUSSION
The data presented in Table 1 show that in mice given a single injection of sheep erythrncytes (10’ in 0.5 ml) and three injections of D&, the number of 19s PFC detected was 2.3-6.8-fold greater than that stimulated by antigen (SE) alone. Tile dose of SE ( 107) was a suboptimal immunizing dose, chosen to ensure observation of any prevailing additive effect. The optimal immutiizing dose was found to be 10” SE/O.5 ml/mouse. The enhancement effect of DBcAMP was not consistently observed with the optimal SE dose. The optimal DBcAMP dose was 0.3 mg/mouse and was effective river at least a lOO-fold concentration range (0.03-3.0 mg) as shnwn in Table I, top group. The enhancement effect prevailed through the late 19s PFC response at two antigen dose levels (10’ and 10” SE/mouse) and the 0.3- and 3.0-mg levels of DBcAMP as shown in Table 1, last two groupings.
SHORT
COMMUNICATIONS
509
The data of Table 2 show that enhancement prevails into the early 7S PFC response. However, this effect could not be discerned ill a late 7S respo”se after 17 . 14l)crirli”iuliization. The data of Table 3 demonstrate DBcAnlP-enhancetl recovery uf mice from the immune defect presumably resulting from neonatal thymectomy (2). In this experiment using 15-20 CIIHf/HeN mice per group, thymectomized, SE-injected control mice mounted only 27% as many PFC as did the sham-thymectomized, SEinjected controls. Whereas, the thymectomized, SE-injected test group, given three l.O-mg injections of DBcAMP retained 7.5% of the control response and exceeded the response of the thymectomized, SE-injectedbut non-DBcAMP-injected group by 2.7-fold. Tabulation of spleen cell counts did not reveal extensive spleen cell proliferation in this study. With the exception of the thyniectoniized, SE-injected, DBcAMPtreated group, the ratio of control to all test group cell counts was essentially equal to 1 (ranged from 0.87 to 1.21). An increase in ceil number of 21 ‘/o in TX, SE, and DBcAMP-injectetl mice (Table 3) may account for part of the DBcAMP-induced recovery of antihotly plaque-forming activity. Tt is possible that the increased cell TABLE INCREASED
EARLY AND LATE MICE INJECTED
1
19s ANTI-SHEEP WITH DIBUTYRYL
19s PFC,‘lO” SPLEEN Experiment number SE.
DBcAM
hfean
1 x 1 x 1 x Three 1 x
107 107 10’
3
C-J 3.0 mg
(--I
0.03 mg 0.3 mg 3.0 mg
II
III
3
IV
f
SEM
Ph
4 -
-
0.20 0.10
Control (-DBcAMP) Control (-SE) 2.3 6.8 4.0
(-DBcAMP) 2.7 2.7
(-DBcAMP) 3.5 2.9
6 16 14
5 15 16
15 50 19
-
-
9 f 2.5 27 zk 10.8 16 f 0.9
15
13
11
11
-
13
irljectionse IO’
(-)
Control
1 x 1 x
10’ 10’
0.3 mg 3.0 mg
36 41
46 32
29 34
28 33
-
35 f 3.7 3.5 r!z 1.4
1 x
108
C-1
31
33
27
31
27
30
1 x 1 x
OF
v
s-<1
RESIWNSES
CELLS
P” I
One irljectk& 1 x 10’
EKYTHROCYTE CYCLIC AMP
10” 108
0.3 mg 3.0 mg
217 106
106 102
67 74
69 75
69 76
106 f 87 f
Control 28.3 6.3
n DBcAMP was injected at these concentrations with SE initially and at two additional times on alternate days. b Prob,lbility of significant differences between the DBcAMP test groups and the (-) DBcAMP control group, calculated by Students’ t test. c PFC/lOG of (f) DBcAMP (f) SE test group PFC/lW of (-) DBcAMP (f) SE control group’ d Responses were measured 4 days later. B Three alternate-day injections, responses were measured
3 days
after
the third.
SlfoRT
510
C’OMMLJNICATTONS TABLE
INCKEASED
EAKLY
7s ANTI-SHEEP INJECTED WITH
2
EKYTHK~CYTE PFC R~~ao~ses DIBUTYKYL CYCLIC AMP
7s PFC/lO6 SrLoEN Experiment number
SE three injections
DBcAM
P
CELLS” Mean
I
II
III
IV
OF MICE
f
SEM
Pb
Fold increasec
v
1 x 10’ 1 x 107 1 x 107
C-1 0.3 mg 3.0 mg
118 194 346
58 128 109
203 344 689
188 306 726
-
142 243 zk 17.0 468 + 113.9
<0.005 <0.05
Control 1.7 3.3
1 x 1 x 1 x
(-) 0.3 mg 3.0 mg
2.51 350 1400
57 135 216
288 818 944
279 761 966
276 761 963
230 565 f 101.0 898 zk 156.7
<0.02
Control 2.5 3.9
108 10” 108
n Assayed 4 days after the third of three alternate-day injections. b Probability of significant differences between the DBcAMP test groups control group, calculated by Students’ t test. c PFC/lOG of test g&p = PFC/lOG
of control
group
and the (-)
DBcAMI’
’
number was due to T-cell proliferation, stemming from possible residual thymus In this cells not removed, which may escape even carefully performed thymcctomy. respect, DNA synthesis and mitotic activity in cells from rat thymus (9) and from human peripheral blood (10) have been stimulated by DBcAMP. However, caution is warranted here with respect to an over- or an underinterpretation of these data, since the functional classes of cells within the thymus appear now to be more complex than previously recognized, e.g., evidence for both an “amplifier cell” and a “suppressor cell” presumably of the thymus has been gained (1 l), and both of these populations appear to be distinct from the T-cell of earlier accepted dogma (1, 2). Whether or not DBcAMP could be affecting amplifiers and suppressors differentially cannot be discerned from the present data. Final resolution of the DBcAMP effect awaits further study, hut it seems clear that a T-cell requirement (1. 2) may be substituted for or a minimal response may be enhanced in mouse anti-SE responses. TAB1.B INCREASED
SE
Sham thymectomized Thymectomized Thymectomized a Average values For each group.
3
ANTI-SE PFC RESPONSES IN NEONATALLY MICE INJECTED WITH DIBUTYRYL CYCLIC
5 x 5 x 5 x for a single
spleen
DBcAMP
106 106 106
in the pooled
1.0 mg c--j 1.0 mg spleen
THYYECTOMIZED AMP
106 Cells/ spleen
19s PFC/ 106 cells
124a 108 150
158 43 118
cell suspensions
made
7c Control
100 (control) 27 75 of 15-20
spleens
SHORT
COiMRfUNICATIONS
511
ACKNOWLEDGMENTS We thank Doctors R. Crouch, C. Lambert, P. Leder, B. Merchant, and S. Stone for criticizing the manuscript. We are grateful to C. Kunkle and L. Williams for expert secretarial preparations.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Mitchell, G. F., and Miller, J. F. ,4. P., Proc. Nut. Acud. Sci. USA 59, 296, 1968. Miller, J.F.A.P., Basten, A., Sprent, J., and Cheers, C., Cell. Zmn~z~?zol.2, 469, 1971. Mitchison, N. A., Eur. J. I~rln~llzol. 1, 18, 1971. Boyd, W. C., “Fundamentals of Immunology.” Wiley, New York, 1966. Ishizuka, M., Gafni, M., and Braun, W., Proc. Sot. Exp. Biol. Med. 134, 963, 1970. Doria, G., Agarossi, G., and Di Pietro, S., J. In~nl~nol. 108, 268, 1972. Hanna, E. E., and Watson, D. W., Proc. Sot. Exp. Biol. Med. 118, 865, 1965. Hanna, E. E., and Merchant, B., J. Z~rzrr~nol. 107, 172, 1971. Macmanus, J. P., and Whitfield, J. F., Ex#. Cell. Rcs. 58, 188, 1969. Hirschhorn, R., Grossman, J., and Weissmann, G., Proc. Sot. Es). Biol. h!Icd. 133, 1361, 1'970. 11. Baker, P. J., Stashak, P. W., Amsbaugh, D. F., Prescott, B., and Barth, R. F., J. TVU?ZUXO~. 1105, 1581, 1970.
of
by Dibutyryl
D. UZUNOVA
Cyclic AMP in Mice
l, 2 AND EDGAR E. HANNA
Molecular Genetics, National Institute of Child Health National Institutes of Health, Brtlwsda, Mtrr~~la~zti Receizvd
Drcetrlber
and Htrwton 20014
Dazrelopnzettt,
26, 1972
Mke injected with NO-2’-O-dibutyryl-adcnosine 3’,5’ monophosphatc (DBcAMP) showed increased anti-sheep erythrocyte (anti-SE) antibody plaque-forming spleen cell (PFC) responses up to 7-fold greater than control mice. The amount of increase was related to the immunizing dose of SE and to the dose of DBcAMP, and it \vas more pronounced in 19s PFC than in 7s PFC. Mice thymectomized (TX) within 16 hr after birth and injected with SE and DBcAMP at 40 days showed a 2.7-fold greater anti-SE PFC response than TX controls injected with SE alone. DBcAMP restored the PFC response of TX mice to 75% of that seen for sham TX, DBcAMPtreated controls. These data suggest that a T cell-B cell interaction is not stringently requil-ed in mouse anti-SE antibody responses ilc vko, since a T &-like cffcct may he substituted or a minimal response can be enhanced with a soluble amplifier such as dihutyryl cyclic AMP.
INTRODUCTION It has been shown (1, 2) that thymectomized mice show a deficit in their antibndy response to injected sheep erythrocytes (SE). Such findings have led to the conclusion that the SE is a thymus-dependent antigen requiring the complex interaction of at least two cell types, a thymus-influenced, nonantibody secreting lymphoid T cell and a bone marrow-derived precursor of the antibody-secreting cell. An interesting, but complex model involving a physical association of T-cells and imhas been proposed for the munogenic carrier, B-cells and haptenic determinants interaction of these two cell types in response to the more stringent, defined synthetic hal,tens (3). This nlotlel is also assumed 1)~ Inany to be applical)le to tnouse anti-SE responses. However, when it is recalled that the heterophile SE antigencomplex is a superior immune, ven in essentially all vertebrates and is active in some subvertebrates (4), it would appear unnecessary for natural selection of such a complex mechanism as proposed in the model. The data presented here complement the results of some investigations (5) and offers a 1,ossible definition for the active factor reported by others (6), to jointly show
that
cell-free
factors
may
anlplify
the
antibody-secreting
potential
of R cells
in the presence of specific antigen directly. The present data do not question the model’s application in tile mechanism of the stringent synthetic imniunogens (3). 1 Recipient of an NIH Fogerty 2 Present address : Department
International Visiting Fellowship. of Pathophysiology, Higher Medical 507
Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
Institute,
Varna,
Bulgaria.
.3JH
SITOR? (‘OMMX~NTChTIoNS AlATERIALS
AND
METHODS
Mice. c57 BI,/6N males, 6-8 weeks old, averaging 25 g, were fIXJill the NIH central animal facility. C;+Hf/HeN iieunatal mice were obtained from pregnant mothers in our laboratory animal room. Pregnant mothers were supplied as above. This strain was used in the thymcctomy experiment because of their good survival and care by mothers after surgery. Thymectomy (Tx) was performed through a small slit in the neck, by air vacuum, followed by sealing the slit with pernlount (Fisher Scientific). Sham thymectomy included the same l)rocetlure without surking out the thymus tissue. The procedure was carried out under a heat lamp and in the presence of mothers, who were themselves painted with the glue, permount. Experimental mice were injected as indicated 40 days later. Iln?nzlnogen. Sheep blood was obtained in acid citrate solution from the NIH Large Animal ,Facility. Washed sheep erythrocyte (SE) suspensions were prepared by repeated washing in pyrogen-free (7) phosphate-buffered saline (PBS). They were diluted to the desired concentration in Pl3S as noted in the data tables. All injections were made intraperitoneally (ip) . URrAM P, N”-2’-O-dibutyryl-adenosine 3’,5’-cyclic phosphate. sodium. 5H,O, A grade, Lot No. 010162, was obtamecl from Calblochem, Fullerton, CA, and maintained desiccated in the cold. For use it was dissolved in pyrogen-free water and diluted as desired in PBS. Injections were by the ip route. Spleen cell suspensions consisted of whole dispersed spleens pooled from 15-20 mice in each experimental group, shown in the tables to follow. They were made by cutting spleens into small pieces and passing the tissue repeatedly through a 44-pm pore size, stainless-steel mesh cell sieve using ice cold Hanks’ BSS, pH 7.4, as the suspending medium as previously detailed (8). A portion of each cell suspension was cryogenically prepared and frozen ( - 1 “/min) in the I&de electronically controlled, liquid nitrogen biological freezer to facilitate repeat of assays as needed. PFC assays were performed as previously detailed (8), except that mouse IgG SEplaques, recorded as 7S plaques, were developed by use of hyperimmune, &sorbed, rabbit anti-mouse 7S imiiiunoglol~ulin and 109, guinea pig complement in PBS containing optimum Ca’+ and hlg”. Cells were couiited in the Autocytometer I I (Fisher Scientific Co., Pittsburgh, PA), electronic cell counter. RESULTS
AND
DISCUSSION
The data presented in Table 1 show that in mice given a single injection of sheep erythrncytes (10’ in 0.5 ml) and three injections of D&, the number of 19s PFC detected was 2.3-6.8-fold greater than that stimulated by antigen (SE) alone. Tile dose of SE ( 107) was a suboptimal immunizing dose, chosen to ensure observation of any prevailing additive effect. The optimal immutiizing dose was found to be 10” SE/O.5 ml/mouse. The enhancement effect of DBcAMP was not consistently observed with the optimal SE dose. The optimal DBcAMP dose was 0.3 mg/mouse and was effective river at least a lOO-fold concentration range (0.03-3.0 mg) as shnwn in Table I, top group. The enhancement effect prevailed through the late 19s PFC response at two antigen dose levels (10’ and 10” SE/mouse) and the 0.3- and 3.0-mg levels of DBcAMP as shown in Table 1, last two groupings.
SHORT
COMMUNICATIONS
509
The data of Table 2 show that enhancement prevails into the early 7S PFC response. However, this effect could not be discerned ill a late 7S respo”se after 17 . 14l)crirli”iuliization. The data of Table 3 demonstrate DBcAnlP-enhancetl recovery uf mice from the immune defect presumably resulting from neonatal thymectomy (2). In this experiment using 15-20 CIIHf/HeN mice per group, thymectomized, SE-injected control mice mounted only 27% as many PFC as did the sham-thymectomized, SEinjected controls. Whereas, the thymectomized, SE-injected test group, given three l.O-mg injections of DBcAMP retained 7.5% of the control response and exceeded the response of the thymectomized, SE-injectedbut non-DBcAMP-injected group by 2.7-fold. Tabulation of spleen cell counts did not reveal extensive spleen cell proliferation in this study. With the exception of the thyniectoniized, SE-injected, DBcAMPtreated group, the ratio of control to all test group cell counts was essentially equal to 1 (ranged from 0.87 to 1.21). An increase in ceil number of 21 ‘/o in TX, SE, and DBcAMP-injectetl mice (Table 3) may account for part of the DBcAMP-induced recovery of antihotly plaque-forming activity. Tt is possible that the increased cell TABLE INCREASED
EARLY AND LATE MICE INJECTED
1
19s ANTI-SHEEP WITH DIBUTYRYL
19s PFC,‘lO” SPLEEN Experiment number SE.
DBcAM
hfean
1 x 1 x 1 x Three 1 x
107 107 10’
3
C-J 3.0 mg
(--I
0.03 mg 0.3 mg 3.0 mg
II
III
3
IV
f
SEM
Ph
4 -
-
0.20 0.10
Control (-DBcAMP) Control (-SE) 2.3 6.8 4.0
(-DBcAMP) 2.7 2.7
(-DBcAMP) 3.5 2.9
6 16 14
5 15 16
15 50 19
-
-
9 f 2.5 27 zk 10.8 16 f 0.9
15
13
11
11
-
13
irljectionse IO’
(-)
Control
1 x 1 x
10’ 10’
0.3 mg 3.0 mg
36 41
46 32
29 34
28 33
-
35 f 3.7 3.5 r!z 1.4
1 x
108
C-1
31
33
27
31
27
30
1 x 1 x
OF
v
s-<1
RESIWNSES
CELLS
P” I
One irljectk& 1 x 10’
EKYTHROCYTE CYCLIC AMP
10” 108
0.3 mg 3.0 mg
217 106
106 102
67 74
69 75
69 76
106 f 87 f
Control 28.3 6.3
n DBcAMP was injected at these concentrations with SE initially and at two additional times on alternate days. b Prob,lbility of significant differences between the DBcAMP test groups and the (-) DBcAMP control group, calculated by Students’ t test. c PFC/lOG of (f) DBcAMP (f) SE test group PFC/lW of (-) DBcAMP (f) SE control group’ d Responses were measured 4 days later. B Three alternate-day injections, responses were measured
3 days
after
the third.
SlfoRT
510
C’OMMLJNICATTONS TABLE
INCKEASED
EAKLY
7s ANTI-SHEEP INJECTED WITH
2
EKYTHK~CYTE PFC R~~ao~ses DIBUTYKYL CYCLIC AMP
7s PFC/lO6 SrLoEN Experiment number
SE three injections
DBcAM
P
CELLS” Mean
I
II
III
IV
OF MICE
f
SEM
Pb
Fold increasec
v
1 x 10’ 1 x 107 1 x 107
C-1 0.3 mg 3.0 mg
118 194 346
58 128 109
203 344 689
188 306 726
-
142 243 zk 17.0 468 + 113.9
<0.005 <0.05
Control 1.7 3.3
1 x 1 x 1 x
(-) 0.3 mg 3.0 mg
2.51 350 1400
57 135 216
288 818 944
279 761 966
276 761 963
230 565 f 101.0 898 zk 156.7
<0.02
Control 2.5 3.9
108 10” 108
n Assayed 4 days after the third of three alternate-day injections. b Probability of significant differences between the DBcAMP test groups control group, calculated by Students’ t test. c PFC/lOG of test g&p = PFC/lOG
of control
group
and the (-)
DBcAMI’
’
number was due to T-cell proliferation, stemming from possible residual thymus In this cells not removed, which may escape even carefully performed thymcctomy. respect, DNA synthesis and mitotic activity in cells from rat thymus (9) and from human peripheral blood (10) have been stimulated by DBcAMP. However, caution is warranted here with respect to an over- or an underinterpretation of these data, since the functional classes of cells within the thymus appear now to be more complex than previously recognized, e.g., evidence for both an “amplifier cell” and a “suppressor cell” presumably of the thymus has been gained (1 l), and both of these populations appear to be distinct from the T-cell of earlier accepted dogma (1, 2). Whether or not DBcAMP could be affecting amplifiers and suppressors differentially cannot be discerned from the present data. Final resolution of the DBcAMP effect awaits further study, hut it seems clear that a T-cell requirement (1. 2) may be substituted for or a minimal response may be enhanced in mouse anti-SE responses. TAB1.B INCREASED
SE
Sham thymectomized Thymectomized Thymectomized a Average values For each group.
3
ANTI-SE PFC RESPONSES IN NEONATALLY MICE INJECTED WITH DIBUTYRYL CYCLIC
5 x 5 x 5 x for a single
spleen
DBcAMP
106 106 106
in the pooled
1.0 mg c--j 1.0 mg spleen
THYYECTOMIZED AMP
106 Cells/ spleen
19s PFC/ 106 cells
124a 108 150
158 43 118
cell suspensions
made
7c Control
100 (control) 27 75 of 15-20
spleens
SHORT
COiMRfUNICATIONS
511
ACKNOWLEDGMENTS We thank Doctors R. Crouch, C. Lambert, P. Leder, B. Merchant, and S. Stone for criticizing the manuscript. We are grateful to C. Kunkle and L. Williams for expert secretarial preparations.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Mitchell, G. F., and Miller, J. F. ,4. P., Proc. Nut. Acud. Sci. USA 59, 296, 1968. Miller, J.F.A.P., Basten, A., Sprent, J., and Cheers, C., Cell. Zmn~z~?zol.2, 469, 1971. Mitchison, N. A., Eur. J. I~rln~llzol. 1, 18, 1971. Boyd, W. C., “Fundamentals of Immunology.” Wiley, New York, 1966. Ishizuka, M., Gafni, M., and Braun, W., Proc. Sot. Exp. Biol. Med. 134, 963, 1970. Doria, G., Agarossi, G., and Di Pietro, S., J. In~nl~nol. 108, 268, 1972. Hanna, E. E., and Watson, D. W., Proc. Sot. Exp. Biol. Med. 118, 865, 1965. Hanna, E. E., and Merchant, B., J. Z~rzrr~nol. 107, 172, 1971. Macmanus, J. P., and Whitfield, J. F., Ex#. Cell. Rcs. 58, 188, 1969. Hirschhorn, R., Grossman, J., and Weissmann, G., Proc. Sot. Es). Biol. h!Icd. 133, 1361, 1'970. 11. Baker, P. J., Stashak, P. W., Amsbaugh, D. F., Prescott, B., and Barth, R. F., J. TVU?ZUXO~. 1105, 1581, 1970.