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Tolerance in early embryo aggregation-derived mouse chimaeras
CELLULAR
IMMUNOLOGY
Tolerance
21, 146-152 (1976)
in Early Embryo Aggregation-Derived
Mouse Chimaeras
R.D. BARNES AND C.G.GRAHAM Clinical
Research
Centre, Harrow, Received
and Department August
of Zoology,
Oxford,
England
26,1975
The concept of tolerance in early embryo aggregation-derived mouse chimaeras has recently been questioned. Here we have looked at the reactivity of chimaera lymphoid cells against corresponding Fl recipients using a local graft-versus-host (GVH) assay. Results in the popliteal lymph node weight gain assay clearly showed diminished reactivity of washed chimaera lymphoid cells against Fl recipients. Although the effect of any serum “blocking” activity cannot, with certainty, be ruled out, evidence clearly points to primary immunological inertia of chimaera lymphoid cells against Fl recipients. In this context it must therefore be assumed that the lymphoid cells from such chimaeras are intrinsically tolerant against parental strain antigens, and the possible mechanisms involved in this process are discussed.
INTRODUCTION Chimaeras can be derived in various ways. One of the most recent methods has involved the aggregation of early embryos (1, 2). Such chimaeras have been referred to as “allophenic” (3) and “tetraparental” (4), but since both terms have limitations the phrase “chimaeras derived by aggregation of early embryos” appears preferable (5). In mice, aggregation of early undifferentiated morulae is best achieved following digestion of the zona pellucida with the enzyme pronase (2). Following successful transplantation into pregnant or preferably pseudopregnant recipients, chimaeras presenting an admixture of the two different parental strain cell populations result. Upon the basis of the persistence of parental strain skin grafts, Mintz and Silvers proposed that such chimaeras were an example of both “permanent and intrinsic tolerance” (3). Both views have since been disputed. While Wegmann and his colleagues have shown that lymphoid cells from such chimaeras can react against parental antigens in vitro (4, 6), we have shown that such an interaction can also occur in viva. In the latter situation graft-versus-host-like activity has been noted (7, 8) and on occasions parental strain skin grafts have been rejected (7, 9). The fact that a serum factor present in the chimaeras “blocks” both cytotoxicity (4) and mixed lymphocyte reactivity (6) in vitro led to the suggestion that tolerance in such chimaeras is generally dependent upon the in vivo activity of the same serum blocking factor (4, 6). Here we have reexamined the situation of tolerance in the chimaeras using a local graft-versus-host (GVH) assay in an attempt to learn whether their lymphoid cells are capable of reacting against corresponding Fl recipients. 146 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
TOLERANCE
MATERIALS
IN
MOUSE
CHIMAEKAS
147
AND METHODS
Mice. Chimaeras were derived by aggregation of early embryos obtained from inbred AKR, CBA/H-T6, C57/Bl, colony-bred PO and naturally derived hybrids. The technique for obtaining such chimaeras has been described in detail previously (7). Naturally derived hybrids were used as recipients in the GVH assay. GVH assay. The local popliteal lymph node weight gain assay originally described by Ford and his colleagues (10) modified for use in the mouse (11) was employed here. Two forms of assay were employed, both in Fl recipients. In the first assay, varying numbers of lymphoid cells from the chimaeras were injected into one rear footpad of a corresponding Fl recipient. Controls in this assay included Fl mice injected with lymphoid cells from one or the other parental strain but also, on occasions, mixtures of both parental strain cells. In each case, cell suspensions were prepared from lymph nodes, washed ( X3) in medium 199, standardised upon viability (trypan blue), and doses adjusted in 199 to unit volume (50 /-J)* In the second assay the effect of pretreatment with chimaera sera was examined. In each case 50 pl of serum was first injected into one footpad and followed 30 min. later by injection of a standard dose of chimaera lymphoid cells into botlz footpads. In a few cases parental strain cells were injected into the corresponding Fl recipient after one or the other footpad had been previously injected with serum from the relevant chimaera. Controls for both assays included determining the cell dose-response curve for the injection of parental strain cells into corresponding Fl recipients. All the recipients used in the GVH assay were aged between 6 and 12 weeks and in each case were killed and examined 7 days after injection, the peak of popliteal lymph node weight gain enlargement in the GVH assay (11). In both the first assay and also the dose-response controls enlargement was expressed as a ratio of injected (I) : noninjected (N) popliteal lymph node weights (11). In the second assay where serum was first injected into one footpad the ratio between the weight of the two lymph nodes was held to reflect the effect of pretreatment with chimaera serum. RESULTS The number and composition of the various chimaeras examined here are shown in Table 1. Results in the popliteal lymph node weight gain assay following the injection of chimaera lymphoid cells are also shown in Table 1. In contrast to the controls (Table 2), minimal lymph node enlargement was seen following the injection of lymphoid cells from the chimaeras; equally important was the fact that enlargement appeared totally unrelated to the number of cells injected (Table 1). Clearly, the chimaeras’ lymphoid cells appear unreactive compared with the parental strain + Fl controls. In the controls no difference was seen in the small number of cases where an admixture of the two parental strains was injected. As can be seen from Table 3, pretreatment with sera from the chimaeras had no effect upon the resultant lymph node enlargement following the injection of parental strain lymphoid cells into 12 corresponding Fl recipients. The same was true for the small group of cases where injection of sera was followed by the injection of chimaera cells. Fourteen assays were performed in this group upon samples
148
BARNESANDCRAIIAM TABLE
1
POPLITEAL LYMPH NODE WEIGHT GAIN ASSAY FOLLOWING INJECTION OF TETRAPARENTAL CHIMAERAS LYMPHOID CELLS Chimaera (donor)
AKR
++ CBA/H-T6
Recipient (host)
(1)
(AKR
X CBA/H-T6)Fl
(2) (3)
(4)
(5)
(6) (7)
(8) (9)
(10)
CBA/H-T6
H CS7/Bl
(1)
H PO
(1)
X PO) c-$ PO
(1)
(2)
Mean
2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0
1.239 1.342 1.220 1.112 1.008 1.361 1.002 1.112 1.373 0.909 1.371 1.400 1.230 1.320 1.219 1.007 1.113 1.472 1.373 1.292 1.484 1.000 1.128 1.173 1.019 1.197 1.370 1.270 1.640 1.320
1.361 1.388 1.568 1.232 1.257 1.380 1.290 1.394 1.362 1.478 1.333 1.321 1.492 1.489 1.327 1.497 1.134 1.148 1.187 1.087 1.274 1.394 1.470
1.380 1.398 1.279 1.559 1.421 1.380 1.379 1.573 1.412 1.640 1.340 1.374 1.487 1.532
1.327 1.376 1.394 1.208 1.275 1.387 1.224 1.253 1.373 1.217 1.371 1.400 1.354 1.327 1.270 1.250 1.113 1.472 1.478 1.344 1.491 1.258 1.203 1.180 1.160 1.233 1.417 1.270 1.640 1.441
X CS7/Bl)Fl
2.5 5.0 10.0 2.5 5.0 10.0
1.009 0.980 1.370 0.808 1.243 1.123
1.373 1.435 1.526 0.993 1.373 1.371
1.578 1.473 1.492 1.398
1.192 1.208 1.491 1.091 1.369 1.297
(CBA/H-T6
x PO)Fl
2.5 5.0 10.0 2.5 5.0 10.0
1.347 1.457 1.430 0.972 1.100 1.123
1.420 1.460 1.472 1.230 1.174
1.473 -
1.384 1.463 1.451 0.972 1.165 1.149
2.5 5.0 10.0 2.5 5.0 10.0
1.473 1.392 1.574 1.323 1.201 1.229
1.498 1.537 1.620 1.420 1.530 1.341
-
1.486 1.465 1.597 1.372 1.366 1.285
(2)
CS7/Bl
Enlargement index (injected/ noninjected)
(CBA/H-T6
(2)
CBA/H-T6
Number of cells injected (X106)
(CS7/Bl
x PO)Fl
TOLERANCE
IN
MOUSE
TABLE
140
CIIIMAERAS
2
POPL~TEAL LYMPH NODE WEIGHT GAIN ASSAY FOLLOWING INJECTIOS OF PARENTAL CELLS INTO F1 HYBRIDS Recipient
Donor
(number
of mice)
Number of cells injected (X 10s)
Cm x CBA/H-T6)Fl
CBA/H-T6J
(32)
CBA/H-T6
(21) (CBA/H-T6
X C57/Bl)Fl
C57/Bl
(19)
CBA/H-T6
(14) (CBA/H-T6
x PO)Fl
PO
2.5 5.0 10.0 2.5 5.0 10.0
2.12 4.17 6.23 1.97 4.03 6.03
f f f f f f
0.63 0.72 0.72 0.32 0.16 0.91
2.5 5.0 10.0 2.5 5.0 1 10.0
2.73 5.17 7.07 2.90 4.95 5.75
f f f f f f
0.98 0.73 1.23 0.68 0.84 2.32
2.5 5.0 10.0 2.5 5.0 1 10.0
1.74 3.04 5.12 1.63 1.98 5.23
f xk f f f f
0.24 0.61 0.73 0.27 1.47 1.07
I
AKR (AKR
Enlargement indw (injected/noninjectcd)
(17)
obtained from seven of the chimaeras (4 AKReCBA/H-TG, 2 CBA/H-Tk C57/Bl and 1 CBA/H-T6tiPO). In each case 5.0 X lo6 chimaera lymphoid cells were injected into each footpad; one previously injected with 50 ~1 of the corresponding serum. Serum injected/noninjected (I/K) ratios ranged from 0.908 to TABLE
3
EFFECT OF THE PRIOR ADMINISTRATION OF CHIMAERA SEKA UPON LOCAL POPLITEAL LYMPH NODE WEIGHT GAIN ASSAM Recipient
Donor
(number
of mice)
AKR (AKR
CBA/H-T6
Number of cells injected (X10”) (4)
5.0 10.0 5.0 10.0 r
1.07 0.97 1.32 1.09
(4)
{ 5.0 10.0 5.0 10.0
1.01 1.07 1.19 0.93
X CBA/H-T6)Fl
i CBA/H-T6
1
C57/Bl
CBA/H-T6 I _. ...-___
(CBA/H-T6
X C57/Bl)Fl
(CBA/H-T6
X PO)Fl
Enlargement index (untreated/sera treated)
0.71 0.98 1.32 1.00
150
BARNES
AND
GRAHAM
1.327 (mean, 1.1270). Clearly the prior administration of chimaera serum did not influence enlargement following the injection of chimaera lymph node cells or reduce the enlargement that was a consequence of the injection of parental strain cells. DISCUSSION Results were unequivocal. In contrast to the controls, very little popliteal lymph node enlargement was noted following the injection of chimaera Iymphoid cells into corresponding Fl recipients. Furthermore, and perhaps more important, any enlargement was totally unrelated to the number of cells injected. Earlier findings noted that injection of nonreactive cells normally caused some slight enlargement in the popliteal lymph node weight gain GVH assay. Injecting 5.0 x 106 Xirradiated, heat-killed and syngeneic (Fl) cells resulted in I/N ratios of 1.167, 1.375 and 1.239 respectively (1 l), ratios that are remarkably similar to the findings here following the injection of the same number of viable chimaera lymphoid cells. Again in contrast to our earlier findings (11) and also the controls here, enlargement following injection of chimaera lymphoid cells appeared totally unrelated to the number of cells injected. Both features point to the fact that, in this assay, lymphoid cells from chimaeras appear immunologically inert. No effect could be attributed to the prior injection of chimaera sera. This is not surprising in the case of the mice subsequently injected with chimaera lymph node cell suspensions, since as noted earlier enlargement here was minimal. However, the fact that sera administered immediately prior to the injection of parental cells failed to prevent any degree of subsequent enlargement, argues for the absence of detectable serum “blocking” activity. However, this assumption is limited since it is only possible to inject a very small volume into the footpad of a mouse at any one time. There were various reasons why we only used one single injection of serum. One obvious reason was the limited quantity of serum available. Second, we were uncertain of the effect of recurrent injections of serum upon the size of the draining lymph node; more important, we were concerned with the possibility of infection since we had earlier shown that injection of infected medium alone resulted in an increased I/N ratio (11). It can therefore be argued that a serum blocking may have been present but was in an insufficient amount to be detected. Also serum blocking activity may have been induced prior to washing. This aside, the fact remains that lymphoid cells from the chimaeras appear relatively inactive in the local GVH assay. In this respect it seems reasonable to consider such cells as intrinsically immunologically inert, within the concept of basic immunological tolerance. This perhaps is not surprising since such chimaeras are commonly obtained by aggregation of very early eight-cell embryos, a perfect situation for immunological self-tolerance to be induced. In contrast to our findings, Wegmann and his associates demonstrated that lymphoid cells from the chimaeras react in vitro in cytotoxicity assays against cells with the corresponding parental strain antigens (4). Again and also in vitro, Phillips and his co-workers showed similar reactivity in mixed lymphocyte culture (6). In both cases the reaction could be blocked by a factor present in the serum of the corresponding chimaera. Our results here clearly conflict with the earlier
TOLERANCE
IN
MOUSE
151
CHIMAERAS
findings. One obvious expltination is that, whereas the earlier work was done in vitro, our assays wer performed in viva. Although this is one obvious explanation, other factors need to be considered. Although we do not dispute the findings of Wegmann and his colleagues (4) we must state that we have failed to confirm their findings. However, this was in what is perhaps considered an atypical situation. Using the same Hellstrom microcytoxicity assay (13) we failed to demonstrate blocking activity in the sera of any of four NZB-CFW chimaeras (12). There were obvious limitations in considering this finding in general in a potentially autoimmune (NZB) situation, particularly since evidence suggested the presence of a GVH-like disease in these chimaeras (7, 8, 12-17). C onceivably, absence of serum blocking activity and GVH-like disease might have been related. It was interesting to note that whereas a hyperactive response in the popliteal lymph node assay was seen in these NZB-CFW chimaeras at a time when there was GVH-like activity (7), a hypoactive response was recorded when GVH-like activity had ceased (17). Therefore one has to conclude that lymphoid cells from the chimaeras are in general unresponsive against Fl recipients, at least in the local popliteal lymph node weight gain assay. The exception is when GVH-like activity occurs. In this context it would seem that the potential for reactivity is present but generally suppressed, a hypothesis that has some support. Phillips recently showed that, in spite of his earlier findings (9), lymphoid cells from the chimaeras do not respond to parental cells in vitro (18). Intriguing is the fact that these same cells are also capable of preventing an interaction between the two corresponding parental strain cell lines. From this work has emerged the possibility of the existence of a suppressor cell population. Presumably inactive in the earlier in vitro studies of Wegmann et al. (4) and Phillips et al. (6) it is this cell population that is manifest in the most recent in vitro studies and also is a likely explanation for the results here. It remains to identify the cell population involved and also the factors that influence its activity, including the possibility of serum-mediated blocking activity. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance Thornton and Mr. Victor Twist in this study.
of Miss
Pamela
Lund,
Miss
Christine
REFERENCES 1. Tarkowski, A. K., Nature (Loxdon) 190, 857, 1961. 2. Mintz, B., Scie+zce 138, 594, 1962. 3. Mintz, B., and Silvers, W. K., Science 158, 1484, 1967. 4. Wegmann, T. G., Hellstrom, I., and Hellstrom. K. E., Proc. Nat. Acad. Sci. 1644, 1971. 5. Barnes, R. D., Iti “Proceedings of 2nd International Conference of Immunology.” 6. Phillips, S. M., Martin, W. J., Shaw, A. R., and Wegmann, T. G., Nntlirc 234, 146, 1971. 7. Barnes, R. D., Tuffrey, M., Kingman, J., Thornton, C., Turner, M. W, C[in. Exp. 11, 605, 1972. 8. Barnes, R. D., Holliday, J., and Tuffrey, M., I~~~mnologg 26, 1195, 1974. 9. Mintz, B., and Palm, J., J. Exp. Med. 129, 1013, 1969. 10. Ford, W. L., Burr, W., and Simonsen, M., Trassplantatiolt 10, 258, 1970. 11. Twist, V., and Barnes, R. D., Transp&fotioti 15, 183, 1973. 12. Barnes, R. D., and Tuffrey, RI., Eur. J. Immunol. 3, 60, 1973.
T‘.C.-l
68,
(f.orxfo)t) 11r11rr~nol.
152
BARNES
AND
GRAHAM
13. Barnes, R. D., and Tuffrey, M., In “Microenvironmental Aspects of Immunity” (B. D. Jankovic and K. Isakovic, Eds.), p. 427. Plenum, New York. 14. Barnes, R. D., and Tuffrey, M., Stand. J. Immunol. 1, 284, 1972. 1.5. Tuffrey, M., Holliday, J., and Barnes, R. D., Pathology 113, 61, 1974. 16. Barnes, R. D., Tuffrey, M., Graham, C. F., Holliday, J., and Thornton, C., Scalzd. J. Zmmunol. 3, 789, 1974. 17. Barnes, R. D., Tuffrey, M., and Wills, E. J., Z. Zmmulzogenet., 2, 19.5, 1975. 18. Phillips, S. M., and Wegmann, T. G., J. Exp. Med. 137, 291, 1973.
IMMUNOLOGY
Tolerance
21, 146-152 (1976)
in Early Embryo Aggregation-Derived
Mouse Chimaeras
R.D. BARNES AND C.G.GRAHAM Clinical
Research
Centre, Harrow, Received
and Department August
of Zoology,
Oxford,
England
26,1975
The concept of tolerance in early embryo aggregation-derived mouse chimaeras has recently been questioned. Here we have looked at the reactivity of chimaera lymphoid cells against corresponding Fl recipients using a local graft-versus-host (GVH) assay. Results in the popliteal lymph node weight gain assay clearly showed diminished reactivity of washed chimaera lymphoid cells against Fl recipients. Although the effect of any serum “blocking” activity cannot, with certainty, be ruled out, evidence clearly points to primary immunological inertia of chimaera lymphoid cells against Fl recipients. In this context it must therefore be assumed that the lymphoid cells from such chimaeras are intrinsically tolerant against parental strain antigens, and the possible mechanisms involved in this process are discussed.
INTRODUCTION Chimaeras can be derived in various ways. One of the most recent methods has involved the aggregation of early embryos (1, 2). Such chimaeras have been referred to as “allophenic” (3) and “tetraparental” (4), but since both terms have limitations the phrase “chimaeras derived by aggregation of early embryos” appears preferable (5). In mice, aggregation of early undifferentiated morulae is best achieved following digestion of the zona pellucida with the enzyme pronase (2). Following successful transplantation into pregnant or preferably pseudopregnant recipients, chimaeras presenting an admixture of the two different parental strain cell populations result. Upon the basis of the persistence of parental strain skin grafts, Mintz and Silvers proposed that such chimaeras were an example of both “permanent and intrinsic tolerance” (3). Both views have since been disputed. While Wegmann and his colleagues have shown that lymphoid cells from such chimaeras can react against parental antigens in vitro (4, 6), we have shown that such an interaction can also occur in viva. In the latter situation graft-versus-host-like activity has been noted (7, 8) and on occasions parental strain skin grafts have been rejected (7, 9). The fact that a serum factor present in the chimaeras “blocks” both cytotoxicity (4) and mixed lymphocyte reactivity (6) in vitro led to the suggestion that tolerance in such chimaeras is generally dependent upon the in vivo activity of the same serum blocking factor (4, 6). Here we have reexamined the situation of tolerance in the chimaeras using a local graft-versus-host (GVH) assay in an attempt to learn whether their lymphoid cells are capable of reacting against corresponding Fl recipients. 146 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
TOLERANCE
MATERIALS
IN
MOUSE
CHIMAEKAS
147
AND METHODS
Mice. Chimaeras were derived by aggregation of early embryos obtained from inbred AKR, CBA/H-T6, C57/Bl, colony-bred PO and naturally derived hybrids. The technique for obtaining such chimaeras has been described in detail previously (7). Naturally derived hybrids were used as recipients in the GVH assay. GVH assay. The local popliteal lymph node weight gain assay originally described by Ford and his colleagues (10) modified for use in the mouse (11) was employed here. Two forms of assay were employed, both in Fl recipients. In the first assay, varying numbers of lymphoid cells from the chimaeras were injected into one rear footpad of a corresponding Fl recipient. Controls in this assay included Fl mice injected with lymphoid cells from one or the other parental strain but also, on occasions, mixtures of both parental strain cells. In each case, cell suspensions were prepared from lymph nodes, washed ( X3) in medium 199, standardised upon viability (trypan blue), and doses adjusted in 199 to unit volume (50 /-J)* In the second assay the effect of pretreatment with chimaera sera was examined. In each case 50 pl of serum was first injected into one footpad and followed 30 min. later by injection of a standard dose of chimaera lymphoid cells into botlz footpads. In a few cases parental strain cells were injected into the corresponding Fl recipient after one or the other footpad had been previously injected with serum from the relevant chimaera. Controls for both assays included determining the cell dose-response curve for the injection of parental strain cells into corresponding Fl recipients. All the recipients used in the GVH assay were aged between 6 and 12 weeks and in each case were killed and examined 7 days after injection, the peak of popliteal lymph node weight gain enlargement in the GVH assay (11). In both the first assay and also the dose-response controls enlargement was expressed as a ratio of injected (I) : noninjected (N) popliteal lymph node weights (11). In the second assay where serum was first injected into one footpad the ratio between the weight of the two lymph nodes was held to reflect the effect of pretreatment with chimaera serum. RESULTS The number and composition of the various chimaeras examined here are shown in Table 1. Results in the popliteal lymph node weight gain assay following the injection of chimaera lymphoid cells are also shown in Table 1. In contrast to the controls (Table 2), minimal lymph node enlargement was seen following the injection of lymphoid cells from the chimaeras; equally important was the fact that enlargement appeared totally unrelated to the number of cells injected (Table 1). Clearly, the chimaeras’ lymphoid cells appear unreactive compared with the parental strain + Fl controls. In the controls no difference was seen in the small number of cases where an admixture of the two parental strains was injected. As can be seen from Table 3, pretreatment with sera from the chimaeras had no effect upon the resultant lymph node enlargement following the injection of parental strain lymphoid cells into 12 corresponding Fl recipients. The same was true for the small group of cases where injection of sera was followed by the injection of chimaera cells. Fourteen assays were performed in this group upon samples
148
BARNESANDCRAIIAM TABLE
1
POPLITEAL LYMPH NODE WEIGHT GAIN ASSAY FOLLOWING INJECTION OF TETRAPARENTAL CHIMAERAS LYMPHOID CELLS Chimaera (donor)
AKR
++ CBA/H-T6
Recipient (host)
(1)
(AKR
X CBA/H-T6)Fl
(2) (3)
(4)
(5)
(6) (7)
(8) (9)
(10)
CBA/H-T6
H CS7/Bl
(1)
H PO
(1)
X PO) c-$ PO
(1)
(2)
Mean
2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0 2.5 5.0 10.0
1.239 1.342 1.220 1.112 1.008 1.361 1.002 1.112 1.373 0.909 1.371 1.400 1.230 1.320 1.219 1.007 1.113 1.472 1.373 1.292 1.484 1.000 1.128 1.173 1.019 1.197 1.370 1.270 1.640 1.320
1.361 1.388 1.568 1.232 1.257 1.380 1.290 1.394 1.362 1.478 1.333 1.321 1.492 1.489 1.327 1.497 1.134 1.148 1.187 1.087 1.274 1.394 1.470
1.380 1.398 1.279 1.559 1.421 1.380 1.379 1.573 1.412 1.640 1.340 1.374 1.487 1.532
1.327 1.376 1.394 1.208 1.275 1.387 1.224 1.253 1.373 1.217 1.371 1.400 1.354 1.327 1.270 1.250 1.113 1.472 1.478 1.344 1.491 1.258 1.203 1.180 1.160 1.233 1.417 1.270 1.640 1.441
X CS7/Bl)Fl
2.5 5.0 10.0 2.5 5.0 10.0
1.009 0.980 1.370 0.808 1.243 1.123
1.373 1.435 1.526 0.993 1.373 1.371
1.578 1.473 1.492 1.398
1.192 1.208 1.491 1.091 1.369 1.297
(CBA/H-T6
x PO)Fl
2.5 5.0 10.0 2.5 5.0 10.0
1.347 1.457 1.430 0.972 1.100 1.123
1.420 1.460 1.472 1.230 1.174
1.473 -
1.384 1.463 1.451 0.972 1.165 1.149
2.5 5.0 10.0 2.5 5.0 10.0
1.473 1.392 1.574 1.323 1.201 1.229
1.498 1.537 1.620 1.420 1.530 1.341
-
1.486 1.465 1.597 1.372 1.366 1.285
(2)
CS7/Bl
Enlargement index (injected/ noninjected)
(CBA/H-T6
(2)
CBA/H-T6
Number of cells injected (X106)
(CS7/Bl
x PO)Fl
TOLERANCE
IN
MOUSE
TABLE
140
CIIIMAERAS
2
POPL~TEAL LYMPH NODE WEIGHT GAIN ASSAY FOLLOWING INJECTIOS OF PARENTAL CELLS INTO F1 HYBRIDS Recipient
Donor
(number
of mice)
Number of cells injected (X 10s)
Cm x CBA/H-T6)Fl
CBA/H-T6J
(32)
CBA/H-T6
(21) (CBA/H-T6
X C57/Bl)Fl
C57/Bl
(19)
CBA/H-T6
(14) (CBA/H-T6
x PO)Fl
PO
2.5 5.0 10.0 2.5 5.0 10.0
2.12 4.17 6.23 1.97 4.03 6.03
f f f f f f
0.63 0.72 0.72 0.32 0.16 0.91
2.5 5.0 10.0 2.5 5.0 1 10.0
2.73 5.17 7.07 2.90 4.95 5.75
f f f f f f
0.98 0.73 1.23 0.68 0.84 2.32
2.5 5.0 10.0 2.5 5.0 1 10.0
1.74 3.04 5.12 1.63 1.98 5.23
f xk f f f f
0.24 0.61 0.73 0.27 1.47 1.07
I
AKR (AKR
Enlargement indw (injected/noninjectcd)
(17)
obtained from seven of the chimaeras (4 AKReCBA/H-TG, 2 CBA/H-Tk C57/Bl and 1 CBA/H-T6tiPO). In each case 5.0 X lo6 chimaera lymphoid cells were injected into each footpad; one previously injected with 50 ~1 of the corresponding serum. Serum injected/noninjected (I/K) ratios ranged from 0.908 to TABLE
3
EFFECT OF THE PRIOR ADMINISTRATION OF CHIMAERA SEKA UPON LOCAL POPLITEAL LYMPH NODE WEIGHT GAIN ASSAM Recipient
Donor
(number
of mice)
AKR (AKR
CBA/H-T6
Number of cells injected (X10”) (4)
5.0 10.0 5.0 10.0 r
1.07 0.97 1.32 1.09
(4)
{ 5.0 10.0 5.0 10.0
1.01 1.07 1.19 0.93
X CBA/H-T6)Fl
i CBA/H-T6
1
C57/Bl
CBA/H-T6 I _. ...-___
(CBA/H-T6
X C57/Bl)Fl
(CBA/H-T6
X PO)Fl
Enlargement index (untreated/sera treated)
0.71 0.98 1.32 1.00
150
BARNES
AND
GRAHAM
1.327 (mean, 1.1270). Clearly the prior administration of chimaera serum did not influence enlargement following the injection of chimaera lymph node cells or reduce the enlargement that was a consequence of the injection of parental strain cells. DISCUSSION Results were unequivocal. In contrast to the controls, very little popliteal lymph node enlargement was noted following the injection of chimaera Iymphoid cells into corresponding Fl recipients. Furthermore, and perhaps more important, any enlargement was totally unrelated to the number of cells injected. Earlier findings noted that injection of nonreactive cells normally caused some slight enlargement in the popliteal lymph node weight gain GVH assay. Injecting 5.0 x 106 Xirradiated, heat-killed and syngeneic (Fl) cells resulted in I/N ratios of 1.167, 1.375 and 1.239 respectively (1 l), ratios that are remarkably similar to the findings here following the injection of the same number of viable chimaera lymphoid cells. Again in contrast to our earlier findings (11) and also the controls here, enlargement following injection of chimaera lymphoid cells appeared totally unrelated to the number of cells injected. Both features point to the fact that, in this assay, lymphoid cells from chimaeras appear immunologically inert. No effect could be attributed to the prior injection of chimaera sera. This is not surprising in the case of the mice subsequently injected with chimaera lymph node cell suspensions, since as noted earlier enlargement here was minimal. However, the fact that sera administered immediately prior to the injection of parental cells failed to prevent any degree of subsequent enlargement, argues for the absence of detectable serum “blocking” activity. However, this assumption is limited since it is only possible to inject a very small volume into the footpad of a mouse at any one time. There were various reasons why we only used one single injection of serum. One obvious reason was the limited quantity of serum available. Second, we were uncertain of the effect of recurrent injections of serum upon the size of the draining lymph node; more important, we were concerned with the possibility of infection since we had earlier shown that injection of infected medium alone resulted in an increased I/N ratio (11). It can therefore be argued that a serum blocking may have been present but was in an insufficient amount to be detected. Also serum blocking activity may have been induced prior to washing. This aside, the fact remains that lymphoid cells from the chimaeras appear relatively inactive in the local GVH assay. In this respect it seems reasonable to consider such cells as intrinsically immunologically inert, within the concept of basic immunological tolerance. This perhaps is not surprising since such chimaeras are commonly obtained by aggregation of very early eight-cell embryos, a perfect situation for immunological self-tolerance to be induced. In contrast to our findings, Wegmann and his associates demonstrated that lymphoid cells from the chimaeras react in vitro in cytotoxicity assays against cells with the corresponding parental strain antigens (4). Again and also in vitro, Phillips and his co-workers showed similar reactivity in mixed lymphocyte culture (6). In both cases the reaction could be blocked by a factor present in the serum of the corresponding chimaera. Our results here clearly conflict with the earlier
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findings. One obvious expltination is that, whereas the earlier work was done in vitro, our assays wer performed in viva. Although this is one obvious explanation, other factors need to be considered. Although we do not dispute the findings of Wegmann and his colleagues (4) we must state that we have failed to confirm their findings. However, this was in what is perhaps considered an atypical situation. Using the same Hellstrom microcytoxicity assay (13) we failed to demonstrate blocking activity in the sera of any of four NZB-CFW chimaeras (12). There were obvious limitations in considering this finding in general in a potentially autoimmune (NZB) situation, particularly since evidence suggested the presence of a GVH-like disease in these chimaeras (7, 8, 12-17). C onceivably, absence of serum blocking activity and GVH-like disease might have been related. It was interesting to note that whereas a hyperactive response in the popliteal lymph node assay was seen in these NZB-CFW chimaeras at a time when there was GVH-like activity (7), a hypoactive response was recorded when GVH-like activity had ceased (17). Therefore one has to conclude that lymphoid cells from the chimaeras are in general unresponsive against Fl recipients, at least in the local popliteal lymph node weight gain assay. The exception is when GVH-like activity occurs. In this context it would seem that the potential for reactivity is present but generally suppressed, a hypothesis that has some support. Phillips recently showed that, in spite of his earlier findings (9), lymphoid cells from the chimaeras do not respond to parental cells in vitro (18). Intriguing is the fact that these same cells are also capable of preventing an interaction between the two corresponding parental strain cell lines. From this work has emerged the possibility of the existence of a suppressor cell population. Presumably inactive in the earlier in vitro studies of Wegmann et al. (4) and Phillips et al. (6) it is this cell population that is manifest in the most recent in vitro studies and also is a likely explanation for the results here. It remains to identify the cell population involved and also the factors that influence its activity, including the possibility of serum-mediated blocking activity. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance Thornton and Mr. Victor Twist in this study.
of Miss
Pamela
Lund,
Miss
Christine
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