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Studies on the mitogen responses of germfree allogeneic chimeras
Studies
on the Mitogen Allogeneic
I. Maturation
JEROME Lhrnd
Laboratory.
A.
Responses Chimeras
of Germfree
of Mitogen Responsiveness Duration of Chimerism MATTINGLY’ University
AND of Notre
Received
July
PHYLLIS
Dame.
Nofrr
M. Dome.
with
WEBB Indicma
46556
26. 1977
Germfree allogeneic bone marrow chimeras (ABMC) were produced by the intravenous injection of approximately 10’ bone marrow cells from DBA/2 mice into lethally irradiated C3H mice. In the germfree state, the ABMC showed no histological signs of graft-versushost reactions (GVHR). and a normal lifespan was attained. Splenic lymphocytes of the germfree ABMC were shown to be unable to respond with DNA synthesis to the T-cell mitogens PHA and Con A, as measured by tritiated-thymidine (‘H-TdR) uptake, but 7 months following bone marrow transplant, they abruptly regained the ability to respond normally to these mitogens. The plaque-forming ceil response to sheep red blood cells irl I,ilw. however. remained very low throughout the lifespan of the ABMC and actually declined at the same time as the mitogen responses returned to normal. It was inferred that helper T cells are not required for T-cell-dependent mitogen responsiveness.
INTRODUCTION T-cell mitogens, especially PHAZ and Con A, have been used extensively during the past decade to evaluate T-cell functions in many animals (l-3). In many cases. the response to these lectins has been used as an indicator of immune competence (4,-F). More recently, the response to different mitogens has been useful as amethod of distinguishing different T-cell subpopulations (6-9). The germfree allogeneic bone marrow chimera (ABMC) is a unique research model displaying many defects in T-cell functions (lo), among them helper T-cell ability toward sheep red blood cells (ll-14), skin graft rejection (14, 15), and memory responses to both sheep red blood cells and alloantigens ( 16). Nevertheless, ABMC have approximately 20% splenic Thy l+ cells, as determined by immunofluorescence (15). They possess fully functional B cells (15, 17) and show little sign of a graft-versus-host reaction (GVHR) if maintained in the germfree state (18. 19). The present study shows that splenic lymphocytes of germfree ABMC are unable to respond to PHA and Con A for a few months after transplantation but reacquire the ability to respond to these mitogens after a long period of chimerism. Even so, a ’ Present address: Department of Pathology, Yale University School of Medicine. 3 IO Cedar Street. New Haven, Connecticut 06510. ’ Abbreviations used: ABMC, allogeneic bone marrow chimera(s); Con A, concanavalin A: FCS, fetal calf serum: 3H-TdR, tritiated thymidine; GVHR, graft-versus-host reaction; ip. intraperitoneal; NMS. normal mouse serum: PFC. plaque-forming cell; PHA, phytohemagglutinin-P: SBMC, syngeneic bone marrow chimera(s); SI, stimulation index: SRBC, sheep red blood cells. 121 0090-1229/78/0102-0121$01.00/0 Copyright Q 1978 by Academic Press. Inc. ,411 rights of reproduction in any form reserved.
122
MATTINGLY
AND
WEBB
deficiency in plaque-forming cell (PFC) production remains throughout the lifespan of the ABMC. This suggests that the helper T cell is not necessary for mitogen responses in mice. MATERIALS Strairls
AND METHODS
of Mice
C3H/He and DBA/2 mice of both sexes, obtained from breeding facilities at Lobund Laboratory, were maintained under germfree conditions in plastic flexible isolators. Mice used as bone marrow donors and also as the irradiated recipients of the bone marrow were always 6-8 weeks old at the time of transplantation. X Irradiatim
The source of X rays was a 260 KVP Picker Therapy X-ray machine operated at 250 kV and 15 mA with a filtration of 1.O mm aluminum and 0.25 mm copper. The rate of exposure was between 37 and 42 R/min. Prospective recipients were irradiated dorsoventrally, with a skin-target distance of 46 cm, in a circular polyethylene restrainer placed inside a specially equipped germfree isolator with no metal parts. A single whole body dose of 1000 R was always administered at the same time of day (20). Bone-Marron*
Transplantation
Donor mice were sacrificed by cervical dislocation and the hind femurs were used as the source of bone marrow. Each recipient was injected intravenously within 20-24 hr after irradiation with 0.5 ml of a pooled bone marrow suspension (approximately 10’ cells) in RPM1 1640 (Microbiological Associates, Bethesda. Maryland). C3H mice injected with DBA/2 bone marrow cells are referred to as allogeneic bone marrow chimeras (ABMC). C3H mice injected with C3H bone marrow cells and DBAI2 mice injected with DBAR bone marrow cells are referred to as syngeneic bone marrow chimeras (SBMC). Cell Culture
u’ith Mitogens
Spleens were gently teased through a 60-mesh stainless-steel screen and the cells were washed twice in cold RPM1 1640 and brought to a final concentration of 10 x IO6 viable cells/ml in RPM1 1640 + 10% fetal calf serum (FCS) (Lot C841212, GIBCO, Grand Island, N.Y .). To each well of a sterile Microtest II cell culture plate (Falcon Plastics, Oxnard, Calif.), .Ol ml of the cell suspension was added along with 0.1 ml of phyohemagglutinin (PHA-P, Wellcome Research Laboratories. Beckenham, England), 0.1 ml of concanavalin A (Con A) (Calbiochem. San Diego, California), or 0.1 ml of RPM1 1640 (21). The final mitogen concentrations in each well were 1.O kg/ml of PHA and 2.5 pg/ml of Con A, previously shown to be optimal in this assay (Mattingly and Webb, unpublished observations). The cells were incubated for 48 hr in a humidified atmosphere of 5% CO, and 9570 air, at 37”C, then pulsed with 0.5 ~1 of [nzethv/-3H]thymidine (3H-TdR) (Schwarz/ Mann, Orangeburg, N. Y.) (1.9 CilmM) an additional 16 hr. The cultures were harvested on a MASH II (Microbiological Associates, Bethesda, Md.). Results are expressed as counts per minute (cpm) or as the stimulation index (SI) = cpm of the stimulated cultures / cpm of the unstimulated cultures.
MITOGEN
Splelric Pl~ryllr-Foanlirlg
RESPOSSES
Cell IPFC)
OF
GERMFREE
(:HIhlER.4S
123
Asscq
Mice were injected with 4 x IO8 sheep erythrocytes (SRBC) (Colorado Serum Co.. Denver, Co.) ip and 5 days later the spleens were removed and the direct PFC assay was performed according to Jerne and Nordin (22). The time of maximum PFC for germfree chimeras was previously shown to be 5 days (12). Results are expressed as PFC/lOh spleen cells. PrcJpcrtwricur c!f‘ Anfiseru
Anti-Thy 1.Z was produced according to the method of Reif and Allen (23) and absorbed repeatedly with liver cells. In acomplement-mediated cytotoxicity assay, it lysed 97% of DBAI2 thymocytes at a I:320 dilution and only 35% of DBAI2 spleen ceils at a I:4 dilution. Anti-C3H serum was produced in DBAi2 mice by the ip injection of 5 x 10’ C3H spleen cells weekly for a period of 6 weeks. Ten days after the last injection, the mice were bled. The resulting antiserum was absorbed with C3H red cells, heat inactivated at 56°C for 30 min, and frozen at -70°C until used. The reverse procedure was performed for anti-DBAi2 serum. These antisera showed greater than 959 specific lysis at a 1:40 dilution, when tested on the spleen cells of the appropriate strain. Pl,c~1,.clrrtt,lrtlt of’ Cells \j.itll Antisrnrm
In each of five separate test tubes, 0.5 ml of a cell suspension (20 x IO6 viable cells/ml) was mixed with 0.1 ml of various dilutions of anti-Thy 1.2, anti-DBAR. anti-C3H, normal mouse serum (NMS), or RPM1 1640. plus 0.2 ml of guinea pig serum ( 1:8) (GIBCO, Grand Island, N.Y .) as a source of complement and incubated for 30 min at 37°C. The cells were then centrifuged, washed in RPM1 1640, resuspended in RPM1 f640 to 1.0 ml, and tested for mitogen responsiveness and PFC ability. RESULTS Normal germfree C3H and DBA/2 showed a rapid increase in the response to PHA and Con A over a 2- to 3-month period, followed by a slow decline with age. These mice generally yielded SI values between 6 and 12 for both mitogens, with both strains showing a slightly higher SI for Con A. Since the SI values were always greater than 6 in the normal mice, this SI value was taken to be the lower normal limit. An SI of less than 6 was considered to be nonresponsive. In syngeneic chimeras (SBMC). responses to PHA and Con A returned to normal within 3 months of bone marrow transplantation and thereafter remained at normal levels (Fig. I). In allogeneic chimeras (ABMC), the PHA and Con A responses remained negligible for 6 months and then rose rapidly above the normal level (Fig. 2). Normal mitogen responses were found in the oldest ABMC tested, which was 17 months after transplantation (results not shown). The PFC response to an ip injection of SRBC (T-cell dependent) in SBMC was similar to that in normal germfree mice of the same strains (Table 1). However, the maximum PFC response was delayed by 24 hr, as previously reported (12). The PFC response in ABMC did not return to normal, regardless of the duration of
124
MATTINGLY
AND
WEBB
20 r
(A)
A
C3H
T
DE812
SSMC SBMC
4 2 : F u)
20
(B)
T
, 4
0 2 MONTHS
AFTER
6 MARROW
,
8 IO TRANSPLANT
, 12
FIG. 1. Stimulation indices (mean + SEM) obtained by testing spleen cells from germfree SBMC of various durations after bone marrow transplantation with mitogens. Range of unstimulated cultures in cpm for C3H SBMC = 6250 + 1400 and for DBA/2 SBMC = 9200 k 1800. Each group represents six or more SBMC. 30
25
r -
l
PHA
T
0 Con A
0 2 MONTHS
4 AFTER
6 MARROW
6
IO TRANSPLANT
12
FIG. 2. Stimulation indices (mean L SEM) obtained by testing spleen cells from germfree ABMC of various durations after bone marrow transplantation with mitogens. ABMC are lethally irradiated DBA/? mice reconstituted with C3H bone marrow cells. Range of unstimulated cultures in cpm = 8700 t 1600. Each group represents six or more ABMC.
MITOCEN
RESPONSES
OF
GERMFREE
TABLE NLMREH
OF
PFCIIW
SPLEEN
3 6 9 12 Months
1472 1789 2117 1132
posttransplant
1391 1179 1401 909 1004
are expressed
ik i k
DBAI2
3 6 8 IO 12 ” Results
1
CELLS
OF I-H~
as mean
PFC
C3H II?” 98 154 204
SBMC ? c k 2 rt
M~c:l.
GERhWRtE
DBAl2
Age (months)
12.5
CHIMERAS
191 11’ 235 64 241
2693 1871 2437 1430
2 i i i
C3H
SBMC
ABMC
2 2 t 2 ?
171 164 142 120 114
2184 1510 2324 1757 1577
200 I05 233 174
128 129 69 113 314
2 i t tt
IO 6 16 IS 16
2 SEM.
chimerism, but remained near 10% of the level observed in SBMC. Similar findings have been reported by others (12, 13, 15, 24). Both PHA and Con A responses were eliminated by prior treatment of the spleen cells with anti-Thy 1.2 plus complement (Table 2). The PFC responses were. of course, unaffected. On the other hand, treatment with anti-H-2 plus complement eliminated homologous responding cells for both types of response in both strains of SBMC and established that the responding cells in the ABMC were of donor (DBA/2) haplotype (Fig. 3). Thus the lymphoid cells of germfree ABMC do not revert to host-type after prolonged chimerism, at a time when mitogen respones have returned to normal. Others have also shown the lack of haplotype conversion in ABMC (17, 24). DISCUSSION Germfree ABMC have previously been shown to be deficient in helper function in response to SRBC (10. 12-15), shown to be caused by lack of sufficient numbers of TABLE
2
No treatment
Chimera
PHA
6 Y I2
7.2 8.8 8.3 7.3 10.3 9.8 10.9 3.2 4.4 9.6 9.0
DBA-SBMC C3H-SBMC C3H-SBMC C3H-SBMC ABMC ABMC
ABMC ABMC
Sl
Months posttransplant
DBA -SBMC DBA -SBMC DBA-SBMC
Anti-Thy
Direct Con 9.7 13.4 II.7 9.8 14.6 13.4 16.4 3.6 6.2 14.7 9.5
A
II47 1284 1006 876 2156 1894 1872 I56 I45 I14 94
SI
PFCIIO”
spleen cells i c t r c + +k C + i-
84 I32 54 98 147 172 76 I? 6 IX I?
I.2 + Complement
PHA 1.3 I.0 I.1 1.0 2.2 1.0 I.8 1.0 I.1 1.4 I .o
Direct
Con I.3 I.? ?.I I.9 1.8 I.6 2.1 1.0 I 2 I ? I13
A
PFC/ IO”
spleen cell5 1341 I456 986 942 2450 1786 2056 I58 196 I73 IO4
2 I i k 2 t k iz 2 2
II2 I36 IIX 104 200 204 243 22 25 I7 I9
126 60 r 6
50
; f 0
40
: 2 5 P P
30
,’
IO
(A)
I
I
20
H
2000
,^
r
(B)
SYNGENEIC DBA/2
I I
MEDIUM
q ANTI- DBA12 q ANTI-C3H q NMS
SYNGENEIC C3H
ALLOGENEIC
FIG. 3. Spleen cell responses ofgermfree chimeras (8 months after bone marrow transplantation) after vitro treatment with anti-DBA/Z serum, anti-C3H, NMS. or RPM1 1640 plus complement. Responses were measured 5 days after ip injection of4 x lo* SRBC. with the SEM being less than 10% of the mean. (A) PHA response in cpm. (B) Direct PFCIIOh spleen cells.
in
helper T cells (16). The present results show that spleen cells of these animals nevertheless respond at normal levels to T-cell mitogens after prolonged chimerism. These cells were shown to be donor derived, and therefore allogeneic to the host thymus. That they were in fact T cells is strongly suggested by anti-Thy 1.2 treatment (Table 2). Piguet and Vassalli (25) showed that B cells of the chimeras could respond to PHA after 4-5 days in culture, provided that T cells were also present. However, only T cells responded after 2-3 days. the interval used in the present experiments. An explanation for the dichotomy between helper function and mitogen responses may be found in Stobo and Paul’s observation that some T cells respond only to Con A, while others respond to both Con A and PHA (6). Kappfer and Marrack (7) and Harwell er 01. (26) suggest that the helper T cell is responsive only to Con A, while other cell types respond to both mitogens. In our results, the PHA and Con A responses showed similar curves. Therefore, it is possible the ABMC responds via the second of these cell types and lacks the first. The response by long-term. but not short-term, ABMC to PHA and Con A suggests two possibilities: the ABMC either (a) has cells which take a long time to mature to mitogen responsiveness, due to the incompatibility between marrow and thymus or other host tissues, or(b) has nonspecific suppressor cells in the spleen of the short-term, but not the long-term, ABMC. The second possibility is supported
MIl’OGES
RESPOSSES
OF
(GERMFREE
C:HlhlER.tS
177
by the work of Urso and Gengozian who have recently shown the presence of large numbers of nonspecific suppressor cells in short-term ABMC (27). Whether the activity of these suppressor cells waned in later months was not tested in theit system. An attempt to relieve suppression by removal of glass-wool adherent cells according to the method of Folch and Waksman (28) was unsuccessful in the ABMC mouse system. It is possible the nonspecific suppressor cell found by Urso and Gengozian is not responsible for the low mitogen respones of the short-term ABMC, or these cells are not glass adherent in this system. Phillips ct trl. (29) showed that mice undergoing a chronic GVHR could not respond well to SRBC. PHA, or Con A. Their system, however, was quite different from ours. and their mice were not germfree. In germfree chimeras involving AKR and DBAE, having the same major histocompatibility difference as in our system. Pollard and Truitt (18) detected no histological evidence of prolonged GVH disease. We also detected no microscopic lesions attributable to a GVHR in the animals used in the present study. We cannot, therefore, attribute lack of responsiveness to a chronic GVHR in the germfree ABMC. We have. therefore, found a T cell in an allogeneic system which matures to Con A and PHA responsiveness after a long period of chimerism. This cell is not a helper cell to T-dependent antigens and its function is otherwise unknown. From these data, it is inferred that bone marrow-derived T-cell precursors can mature to mitogen responsive T cells in an allogeneic environment, and that helper T cells are unnecessary in a mitogenic response. REFERENCES I ’ i: 4, 5. 6. 7. 8. Y. 10. I I. 12. 13. 14. 15. 16. 17. IX. 19. 20. 21. 22. 23.
Nowell. P. C.. C‘orrc,e,. Rc.s. 20, 462. 1960. Powell. A. E.. and Leon, M. A.. .&[I. Ce//. Rrs. 62, 315, 1970. Stobo. J. I).. 7‘r
128 24. 25. 26. 27. 28. 29.
MATTINGLY
AND
WEBB
Gengozian. N., Congdon, C. C.. and Allen. E. A.. Trtr,~.s/~kurr. Pooch. 3, 434, 1971. Piguet. P. -F., and Vassalli, P., J. E*p. Med. 136, 962. 1972. Harwell. L.. Kappler, J. W., and Marrack. P.. J. Imurr~uol. 116, 1379. 1976. Urso. P., and Gengozian, N.. FCC/. Pmt. (Abst.) 36, 1269, 1977. Folch. H.. and Waksman. B. H., J. Inrm~ruol. 113. 127. 1974. Phillips. S. M.. Gleichmann. H.. Hirsch, M. S.. Black. P.. Merrill, J. P.. Schwartz. Carpenter. C. B.. Cc,//. 1uut1u~101. IS, IS?, 1975.
R. S.. and
on the Mitogen Allogeneic
I. Maturation
JEROME Lhrnd
Laboratory.
A.
Responses Chimeras
of Germfree
of Mitogen Responsiveness Duration of Chimerism MATTINGLY’ University
AND of Notre
Received
July
PHYLLIS
Dame.
Nofrr
M. Dome.
with
WEBB Indicma
46556
26. 1977
Germfree allogeneic bone marrow chimeras (ABMC) were produced by the intravenous injection of approximately 10’ bone marrow cells from DBA/2 mice into lethally irradiated C3H mice. In the germfree state, the ABMC showed no histological signs of graft-versushost reactions (GVHR). and a normal lifespan was attained. Splenic lymphocytes of the germfree ABMC were shown to be unable to respond with DNA synthesis to the T-cell mitogens PHA and Con A, as measured by tritiated-thymidine (‘H-TdR) uptake, but 7 months following bone marrow transplant, they abruptly regained the ability to respond normally to these mitogens. The plaque-forming ceil response to sheep red blood cells irl I,ilw. however. remained very low throughout the lifespan of the ABMC and actually declined at the same time as the mitogen responses returned to normal. It was inferred that helper T cells are not required for T-cell-dependent mitogen responsiveness.
INTRODUCTION T-cell mitogens, especially PHAZ and Con A, have been used extensively during the past decade to evaluate T-cell functions in many animals (l-3). In many cases. the response to these lectins has been used as an indicator of immune competence (4,-F). More recently, the response to different mitogens has been useful as amethod of distinguishing different T-cell subpopulations (6-9). The germfree allogeneic bone marrow chimera (ABMC) is a unique research model displaying many defects in T-cell functions (lo), among them helper T-cell ability toward sheep red blood cells (ll-14), skin graft rejection (14, 15), and memory responses to both sheep red blood cells and alloantigens ( 16). Nevertheless, ABMC have approximately 20% splenic Thy l+ cells, as determined by immunofluorescence (15). They possess fully functional B cells (15, 17) and show little sign of a graft-versus-host reaction (GVHR) if maintained in the germfree state (18. 19). The present study shows that splenic lymphocytes of germfree ABMC are unable to respond to PHA and Con A for a few months after transplantation but reacquire the ability to respond to these mitogens after a long period of chimerism. Even so, a ’ Present address: Department of Pathology, Yale University School of Medicine. 3 IO Cedar Street. New Haven, Connecticut 06510. ’ Abbreviations used: ABMC, allogeneic bone marrow chimera(s); Con A, concanavalin A: FCS, fetal calf serum: 3H-TdR, tritiated thymidine; GVHR, graft-versus-host reaction; ip. intraperitoneal; NMS. normal mouse serum: PFC. plaque-forming cell; PHA, phytohemagglutinin-P: SBMC, syngeneic bone marrow chimera(s); SI, stimulation index: SRBC, sheep red blood cells. 121 0090-1229/78/0102-0121$01.00/0 Copyright Q 1978 by Academic Press. Inc. ,411 rights of reproduction in any form reserved.
122
MATTINGLY
AND
WEBB
deficiency in plaque-forming cell (PFC) production remains throughout the lifespan of the ABMC. This suggests that the helper T cell is not necessary for mitogen responses in mice. MATERIALS Strairls
AND METHODS
of Mice
C3H/He and DBA/2 mice of both sexes, obtained from breeding facilities at Lobund Laboratory, were maintained under germfree conditions in plastic flexible isolators. Mice used as bone marrow donors and also as the irradiated recipients of the bone marrow were always 6-8 weeks old at the time of transplantation. X Irradiatim
The source of X rays was a 260 KVP Picker Therapy X-ray machine operated at 250 kV and 15 mA with a filtration of 1.O mm aluminum and 0.25 mm copper. The rate of exposure was between 37 and 42 R/min. Prospective recipients were irradiated dorsoventrally, with a skin-target distance of 46 cm, in a circular polyethylene restrainer placed inside a specially equipped germfree isolator with no metal parts. A single whole body dose of 1000 R was always administered at the same time of day (20). Bone-Marron*
Transplantation
Donor mice were sacrificed by cervical dislocation and the hind femurs were used as the source of bone marrow. Each recipient was injected intravenously within 20-24 hr after irradiation with 0.5 ml of a pooled bone marrow suspension (approximately 10’ cells) in RPM1 1640 (Microbiological Associates, Bethesda. Maryland). C3H mice injected with DBA/2 bone marrow cells are referred to as allogeneic bone marrow chimeras (ABMC). C3H mice injected with C3H bone marrow cells and DBAI2 mice injected with DBAR bone marrow cells are referred to as syngeneic bone marrow chimeras (SBMC). Cell Culture
u’ith Mitogens
Spleens were gently teased through a 60-mesh stainless-steel screen and the cells were washed twice in cold RPM1 1640 and brought to a final concentration of 10 x IO6 viable cells/ml in RPM1 1640 + 10% fetal calf serum (FCS) (Lot C841212, GIBCO, Grand Island, N.Y .). To each well of a sterile Microtest II cell culture plate (Falcon Plastics, Oxnard, Calif.), .Ol ml of the cell suspension was added along with 0.1 ml of phyohemagglutinin (PHA-P, Wellcome Research Laboratories. Beckenham, England), 0.1 ml of concanavalin A (Con A) (Calbiochem. San Diego, California), or 0.1 ml of RPM1 1640 (21). The final mitogen concentrations in each well were 1.O kg/ml of PHA and 2.5 pg/ml of Con A, previously shown to be optimal in this assay (Mattingly and Webb, unpublished observations). The cells were incubated for 48 hr in a humidified atmosphere of 5% CO, and 9570 air, at 37”C, then pulsed with 0.5 ~1 of [nzethv/-3H]thymidine (3H-TdR) (Schwarz/ Mann, Orangeburg, N. Y.) (1.9 CilmM) an additional 16 hr. The cultures were harvested on a MASH II (Microbiological Associates, Bethesda, Md.). Results are expressed as counts per minute (cpm) or as the stimulation index (SI) = cpm of the stimulated cultures / cpm of the unstimulated cultures.
MITOGEN
Splelric Pl~ryllr-Foanlirlg
RESPOSSES
Cell IPFC)
OF
GERMFREE
(:HIhlER.4S
123
Asscq
Mice were injected with 4 x IO8 sheep erythrocytes (SRBC) (Colorado Serum Co.. Denver, Co.) ip and 5 days later the spleens were removed and the direct PFC assay was performed according to Jerne and Nordin (22). The time of maximum PFC for germfree chimeras was previously shown to be 5 days (12). Results are expressed as PFC/lOh spleen cells. PrcJpcrtwricur c!f‘ Anfiseru
Anti-Thy 1.Z was produced according to the method of Reif and Allen (23) and absorbed repeatedly with liver cells. In acomplement-mediated cytotoxicity assay, it lysed 97% of DBAI2 thymocytes at a I:320 dilution and only 35% of DBAI2 spleen ceils at a I:4 dilution. Anti-C3H serum was produced in DBAi2 mice by the ip injection of 5 x 10’ C3H spleen cells weekly for a period of 6 weeks. Ten days after the last injection, the mice were bled. The resulting antiserum was absorbed with C3H red cells, heat inactivated at 56°C for 30 min, and frozen at -70°C until used. The reverse procedure was performed for anti-DBAi2 serum. These antisera showed greater than 959 specific lysis at a 1:40 dilution, when tested on the spleen cells of the appropriate strain. Pl,c~1,.clrrtt,lrtlt of’ Cells \j.itll Antisrnrm
In each of five separate test tubes, 0.5 ml of a cell suspension (20 x IO6 viable cells/ml) was mixed with 0.1 ml of various dilutions of anti-Thy 1.2, anti-DBAR. anti-C3H, normal mouse serum (NMS), or RPM1 1640. plus 0.2 ml of guinea pig serum ( 1:8) (GIBCO, Grand Island, N.Y .) as a source of complement and incubated for 30 min at 37°C. The cells were then centrifuged, washed in RPM1 1640, resuspended in RPM1 f640 to 1.0 ml, and tested for mitogen responsiveness and PFC ability. RESULTS Normal germfree C3H and DBA/2 showed a rapid increase in the response to PHA and Con A over a 2- to 3-month period, followed by a slow decline with age. These mice generally yielded SI values between 6 and 12 for both mitogens, with both strains showing a slightly higher SI for Con A. Since the SI values were always greater than 6 in the normal mice, this SI value was taken to be the lower normal limit. An SI of less than 6 was considered to be nonresponsive. In syngeneic chimeras (SBMC). responses to PHA and Con A returned to normal within 3 months of bone marrow transplantation and thereafter remained at normal levels (Fig. I). In allogeneic chimeras (ABMC), the PHA and Con A responses remained negligible for 6 months and then rose rapidly above the normal level (Fig. 2). Normal mitogen responses were found in the oldest ABMC tested, which was 17 months after transplantation (results not shown). The PFC response to an ip injection of SRBC (T-cell dependent) in SBMC was similar to that in normal germfree mice of the same strains (Table 1). However, the maximum PFC response was delayed by 24 hr, as previously reported (12). The PFC response in ABMC did not return to normal, regardless of the duration of
124
MATTINGLY
AND
WEBB
20 r
(A)
A
C3H
T
DE812
SSMC SBMC
4 2 : F u)
20
(B)
T
, 4
0 2 MONTHS
AFTER
6 MARROW
,
8 IO TRANSPLANT
, 12
FIG. 1. Stimulation indices (mean + SEM) obtained by testing spleen cells from germfree SBMC of various durations after bone marrow transplantation with mitogens. Range of unstimulated cultures in cpm for C3H SBMC = 6250 + 1400 and for DBA/2 SBMC = 9200 k 1800. Each group represents six or more SBMC. 30
25
r -
l
PHA
T
0 Con A
0 2 MONTHS
4 AFTER
6 MARROW
6
IO TRANSPLANT
12
FIG. 2. Stimulation indices (mean L SEM) obtained by testing spleen cells from germfree ABMC of various durations after bone marrow transplantation with mitogens. ABMC are lethally irradiated DBA/? mice reconstituted with C3H bone marrow cells. Range of unstimulated cultures in cpm = 8700 t 1600. Each group represents six or more ABMC.
MITOCEN
RESPONSES
OF
GERMFREE
TABLE NLMREH
OF
PFCIIW
SPLEEN
3 6 9 12 Months
1472 1789 2117 1132
posttransplant
1391 1179 1401 909 1004
are expressed
ik i k
DBAI2
3 6 8 IO 12 ” Results
1
CELLS
OF I-H~
as mean
PFC
C3H II?” 98 154 204
SBMC ? c k 2 rt
M~c:l.
GERhWRtE
DBAl2
Age (months)
12.5
CHIMERAS
191 11’ 235 64 241
2693 1871 2437 1430
2 i i i
C3H
SBMC
ABMC
2 2 t 2 ?
171 164 142 120 114
2184 1510 2324 1757 1577
200 I05 233 174
128 129 69 113 314
2 i t tt
IO 6 16 IS 16
2 SEM.
chimerism, but remained near 10% of the level observed in SBMC. Similar findings have been reported by others (12, 13, 15, 24). Both PHA and Con A responses were eliminated by prior treatment of the spleen cells with anti-Thy 1.2 plus complement (Table 2). The PFC responses were. of course, unaffected. On the other hand, treatment with anti-H-2 plus complement eliminated homologous responding cells for both types of response in both strains of SBMC and established that the responding cells in the ABMC were of donor (DBA/2) haplotype (Fig. 3). Thus the lymphoid cells of germfree ABMC do not revert to host-type after prolonged chimerism, at a time when mitogen respones have returned to normal. Others have also shown the lack of haplotype conversion in ABMC (17, 24). DISCUSSION Germfree ABMC have previously been shown to be deficient in helper function in response to SRBC (10. 12-15), shown to be caused by lack of sufficient numbers of TABLE
2
No treatment
Chimera
PHA
6 Y I2
7.2 8.8 8.3 7.3 10.3 9.8 10.9 3.2 4.4 9.6 9.0
DBA-SBMC C3H-SBMC C3H-SBMC C3H-SBMC ABMC ABMC
ABMC ABMC
Sl
Months posttransplant
DBA -SBMC DBA -SBMC DBA-SBMC
Anti-Thy
Direct Con 9.7 13.4 II.7 9.8 14.6 13.4 16.4 3.6 6.2 14.7 9.5
A
II47 1284 1006 876 2156 1894 1872 I56 I45 I14 94
SI
PFCIIO”
spleen cells i c t r c + +k C + i-
84 I32 54 98 147 172 76 I? 6 IX I?
I.2 + Complement
PHA 1.3 I.0 I.1 1.0 2.2 1.0 I.8 1.0 I.1 1.4 I .o
Direct
Con I.3 I.? ?.I I.9 1.8 I.6 2.1 1.0 I 2 I ? I13
A
PFC/ IO”
spleen cell5 1341 I456 986 942 2450 1786 2056 I58 196 I73 IO4
2 I i k 2 t k iz 2 2
II2 I36 IIX 104 200 204 243 22 25 I7 I9
126 60 r 6
50
; f 0
40
: 2 5 P P
30
,’
IO
(A)
I
I
20
H
2000
,^
r
(B)
SYNGENEIC DBA/2
I I
MEDIUM
q ANTI- DBA12 q ANTI-C3H q NMS
SYNGENEIC C3H
ALLOGENEIC
FIG. 3. Spleen cell responses ofgermfree chimeras (8 months after bone marrow transplantation) after vitro treatment with anti-DBA/Z serum, anti-C3H, NMS. or RPM1 1640 plus complement. Responses were measured 5 days after ip injection of4 x lo* SRBC. with the SEM being less than 10% of the mean. (A) PHA response in cpm. (B) Direct PFCIIOh spleen cells.
in
helper T cells (16). The present results show that spleen cells of these animals nevertheless respond at normal levels to T-cell mitogens after prolonged chimerism. These cells were shown to be donor derived, and therefore allogeneic to the host thymus. That they were in fact T cells is strongly suggested by anti-Thy 1.2 treatment (Table 2). Piguet and Vassalli (25) showed that B cells of the chimeras could respond to PHA after 4-5 days in culture, provided that T cells were also present. However, only T cells responded after 2-3 days. the interval used in the present experiments. An explanation for the dichotomy between helper function and mitogen responses may be found in Stobo and Paul’s observation that some T cells respond only to Con A, while others respond to both Con A and PHA (6). Kappfer and Marrack (7) and Harwell er 01. (26) suggest that the helper T cell is responsive only to Con A, while other cell types respond to both mitogens. In our results, the PHA and Con A responses showed similar curves. Therefore, it is possible the ABMC responds via the second of these cell types and lacks the first. The response by long-term. but not short-term, ABMC to PHA and Con A suggests two possibilities: the ABMC either (a) has cells which take a long time to mature to mitogen responsiveness, due to the incompatibility between marrow and thymus or other host tissues, or(b) has nonspecific suppressor cells in the spleen of the short-term, but not the long-term, ABMC. The second possibility is supported
MIl’OGES
RESPOSSES
OF
(GERMFREE
C:HlhlER.tS
177
by the work of Urso and Gengozian who have recently shown the presence of large numbers of nonspecific suppressor cells in short-term ABMC (27). Whether the activity of these suppressor cells waned in later months was not tested in theit system. An attempt to relieve suppression by removal of glass-wool adherent cells according to the method of Folch and Waksman (28) was unsuccessful in the ABMC mouse system. It is possible the nonspecific suppressor cell found by Urso and Gengozian is not responsible for the low mitogen respones of the short-term ABMC, or these cells are not glass adherent in this system. Phillips ct trl. (29) showed that mice undergoing a chronic GVHR could not respond well to SRBC. PHA, or Con A. Their system, however, was quite different from ours. and their mice were not germfree. In germfree chimeras involving AKR and DBAE, having the same major histocompatibility difference as in our system. Pollard and Truitt (18) detected no histological evidence of prolonged GVH disease. We also detected no microscopic lesions attributable to a GVHR in the animals used in the present study. We cannot, therefore, attribute lack of responsiveness to a chronic GVHR in the germfree ABMC. We have. therefore, found a T cell in an allogeneic system which matures to Con A and PHA responsiveness after a long period of chimerism. This cell is not a helper cell to T-dependent antigens and its function is otherwise unknown. From these data, it is inferred that bone marrow-derived T-cell precursors can mature to mitogen responsive T cells in an allogeneic environment, and that helper T cells are unnecessary in a mitogenic response. REFERENCES I ’ i: 4, 5. 6. 7. 8. Y. 10. I I. 12. 13. 14. 15. 16. 17. IX. 19. 20. 21. 22. 23.
Nowell. P. C.. C‘orrc,e,. Rc.s. 20, 462. 1960. Powell. A. E.. and Leon, M. A.. .&[I. Ce//. Rrs. 62, 315, 1970. Stobo. J. I).. 7‘r
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MATTINGLY
AND
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Gengozian. N., Congdon, C. C.. and Allen. E. A.. Trtr,~.s/~kurr. Pooch. 3, 434, 1971. Piguet. P. -F., and Vassalli, P., J. E*p. Med. 136, 962. 1972. Harwell. L.. Kappler, J. W., and Marrack. P.. J. Imurr~uol. 116, 1379. 1976. Urso. P., and Gengozian, N.. FCC/. Pmt. (Abst.) 36, 1269, 1977. Folch. H.. and Waksman. B. H., J. Inrm~ruol. 113. 127. 1974. Phillips. S. M.. Gleichmann. H.. Hirsch, M. S.. Black. P.. Merrill, J. P.. Schwartz. Carpenter. C. B.. Cc,//. 1uut1u~101. IS, IS?, 1975.
R. S.. and