CXI.~.~II.AI< III
~I~~.\'oI.oGY
Lymphocytes
25, 189-196 (1976)
Which
Differentiate
I. Response to MLC Determinants
in an Allogeneic
and Skin Grafts
from the Thymus
Thymus Donor Strain
B. KINDKEI) Basrl Imtitu
tc for Imntunology,
Grewacherstrassc Rcceiz’cd Fcbrrlaq
387,Postfach, 400. Rasel 5, Szvit~c~rlartd 18, 1976
INTKODGCTION When a nude mouse is grafted with a neonatal allogeneic thymus, host stem cells migrate into the graft and proliferate. From ahout 25 days after grafting it becomes difficult to find donor type lymphocytes in the graft (l-3) although a population of donor type lymphocytes does appear to persist and is more apparent if mice are examined some months after thymus grafting (4). \\‘hile the change from donor to host type cells is taking place in the thymus, cells also lcave tile and repopulate the peripheral lymphoid organs (1, 2, 5). Some donor tyl)e thymus T cells reach the periphery but the majority of the cells are of host origin (1 ). Tile immune responses in these animals are poorer than in normal mice but are comparable to those of nude mice with congenic thymus grafts (6, 7). These mice provide excellent material for studying the effects of the thymus on lymphocyte differentiation and particularly on the role of the thymus in recognition of self and non-self. From a theory proposed 1~. -Terne (a), one would predict that cells which differentiate in an allogeneic thymus cvould fail to react against tissues of the thymus donor strain. Pritchard and Rlicklem (3) found that this was the case for skin grafts but Seeger et al., (9) report the opposite for heart grafts and Kindred and Loor (6, 7) showed that skin grafts are sometimes accepted and sometimes rejected. The work presented here is a more thorough investigation of this problem and suggests that the animals are indeed tolerant to histocompatibilit~ antigens of the thymus donor strain but may respond to other, perhaps tissue specific, antigens. MATERIALS
AND r\lETHODS
11~01tscstvaim The strains used were BALB/cAnNIcrKb, C57BL/GNIcrKb, C&4/J, AKK/J, 129/J, A/J BALB/c-nunu and CS/“BL/G- 11ullu purchased from Gl. BomholtgSrd and a BALE/c-nunu stock made by direct backcrossing to BALB/cAnNIcrKb. These last were used only to establish that BALB/c-nunu grafted with a tllymus from the same sub-strain did not reject skin from that substrain. The A-TL- strain was derived from a cross-over (Boyse ef al., IO). They were kindly given to us by Dr. Donald Shreffler. 189 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
190 Mixed Lymphocyte
B.
KINDRED
Culture
Lymph node cells were prepared under sterile conditions in Falcon plastic tubes, washed twice and diluted to 107/ml. Target cells were irradiated at 3300 r and 106 responders + lo6 target cells were incubated in each well of a microtiter plate. The medium used was RPM1 1640 medium (Microbiological Associates Inc., Bethesda, Md.), 10% fetal calf serum (Gibco) 0.03 A4 Hepes, and 2 mM glutamin, 100 III/ml of penicillin-streptomycin (Flow Laboratories Ltd., Irvine, Scotland). After 72 hr culture [“HI thymidine was added (2 &i/culture) and the cells were kept in culture for a further 24 hr then harvested, collected on Millipore filters and incubated overnight at 37°C in scintillation vials with 0.5 ml Protosol. The incorporated radioactivity was then measured. Skin Grafting Full thickness skin grafts were made as described by Ballantyne and Converse ( 11). Plasters were removed after 12 days and the grafts were examined daily for 2 weeks after which they were examined twice weekly. Histology Thymuses for histology were fixed in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. The preparation and examination was kindly performed by Dr. J$rgen Rygaard. RESULTS In all the experiments presented here the nude mice were left for at least 5 weeks after thymus grafting to allow ample time for host ‘type precursor cells to enter the thymus, differentiate and repopulate the peripheral lymphoid organs. Mixed Lymphocyte Reaction The ability of cells from BALB/c-nunu with BALB/c (H-2d) or allogeneic thymus grafts to react to the MLR determinants of the major histocompatibility complex was tested. The results are shown in Table 1. Mice with congenic, BALB/c, thymus grafts react equally strongly to C57BL/6J (H-2b) or AKR ( H-2k) and somewhat less strongly to CBA(H-2k). However, in general, mice with allogeneic thymus grafts react very poorly, if at all, to the donor strain or to strains having the same H-2 complex. The low responses may be real but they might also be due to “back stimulation” by the irradiated target cell,s as found by von Boehmer (12). There is one striking exception. The last of the mice listed with an AKR thymus graft reacted strongly to AKR cells. All the mice which produced a stimulation of five times or better against some target have been included in Table 1. However, these were by no means all the animals tested. About half the mice showed very feeble stimulation to all the targets. In some cases the background stimulation incorporation was very high and little increase was possible but this was not always so. There was no apparent correlation with age, status of skin grafts, presence of thymus graft or any other variable we could think of.
RESPONSE
OF GRAFTED
NUDES
TABLE Stimulation Grafts Given Incorporation Thymes
in Mixed Lymphocyte Cultures as [zH]thymidine Incorporation by Responder Cells + Irradiated
TO
I)OSOH
1
by Cells from BALB/c-nrmu Bearing Thymr~s by Responder Cells + Irradiated Target Cells: Responder Cells Target
graft AKR (H-2k)
CBA4(H-2k)
C57BL/6(H-2”)
3.7 6.0 5.2 4.2
5.6 7.6 7.6 11.0
5.80 6.W’ 3.8 9.7 6.1
5.1 10.0 2.4
2.4 1.8 1.0 2.2 0.9
CBA4
4.7
2.-t
11.3
AKR
2.8 2.4 21.3c
2.1 1.6 0.0
11.5 6.6 9.5 ___~~--
BALB,‘c
C57BL/6
101
ANTIGEXS
5.9 8.4 9.7 11.0
a This animal had accepted a C57BL/6 skin graft. h This animal had rejected a C57BL/6 skin graft. Tested in the same experiment c See text for further comment.
129(11-Pj
I .o
as ‘l.
The results of skin grafting experiments (Table 2) are more difficult to interpret than those of mixed lymphocyte cultures. Mice with congenic thymus grafts reject skin grafts from other strains. The rejection is slower than is found with normal mice but grafits are always rejected and second grafts are rejected more rapidly. This is also the case in recipients of allogeneic thymus grafts if they are grafted with skin from a third strain but the pattern of rejection of skin grafts from the thymus donor strain is very unusual. With C57BL/6, CBA or 129 donors half or more of the first ‘skin grafts are rejected but when animals which have rejected grafts are regrafted the second grafts are accepted. However, mice bearing AKR thymus grafts rarely accepted AKR skin grafts even if grafted two or three times. It is an interesting point that acceptance or rejection of skin grafts had no apparent effect on the behaviour of lymphocytes in mixed lymphocyte reaction e.g. the first and second C57BL/6 recipients listed in Table 1 were tested in MLC in the same experiment. Both had been grafted with C57BL/G skin; the first accepted the skin graft and the second rejected it but their reactions in MLC were very similar. As it has not been demonstrated that all donor type thymus cells are lost from a grafted thymus, it seemed possible that the differences in rejection patterns hetweerl CBA and C57BL/6J on the one hand and AKK on the other might be clue to differences in survival of a small but important residual population of donor thymus cells. To test this, BALB/c-nunu were grafted with AKR x C57BL/6 F, thymuses so that any residual population of donor thymocytes would bear both C57BL/6 and AKK histocompatibility antigens. Six such mice were given both C57BL/6
192
B.
KINDRED
TABLE Rejection
Thymus
graft
Skin graft
Rejection 10-15
BALB/c
C57BL/6
time in days
15-20
20-25 2
C57BL/6(lst) C57BL/6(2nd) CBA (1st) CBA (2nd) BALB/c
5 7 4 3
2
C57BL/6(lst) C57BL/6(2nd)b CBA (1st) CBA (2nd)
16
7
6
3 5
2
5
to
Not rejected” (survival 25 + days)
25+ 3
0
1 3(l) 14(6)
869
C57BL/6 (1st) C57BL/6(2nd) CBA (1st) CBA (2nd) AKR (1st) AKR (2nd)b AKR (3rd)” )
1 1 1 2 3 3 1
CBA
CBA CBA
(1st) (2nd) b
4
129
129 129
(1st) (2nd) b
1
AKR
2
Times for Skin Grafts from Various Strains BALB/c-nunu Bearing Thymus Grafts
1 1
1 2
1
2 3
4 1
2
l(l) l(l)
1 5 (2) 4(2) 1
1
1
3(2) 3
a The number in brackets is the number surviving with intact graft for more than 60 days. b 2nd and 3rd grafts were on mice which had rejected the 1st graft.
and AKR sskin grafts side by side. The rejection times for these grafts are shown in Table 3. The AKR grafts were all rejected but about half of the C57BL/6 grafts were accepted. Therefore differences in survival of C57BL/6 and AKR thymocytes cannot be responsible for the differences in pattern of skin graft rejection. Although nudes bearing an F1 thymus reject AKR skin they do not normally reject F1 skin. Six nudes were grafted with AKR x C57BL/6 ,thymuses and later with AKR x C57BL/6 skin. Only one of the skin grafts was rejected after 17 days. The survival times of the other mice with intact grafts were 21, 34, 44, 75 and 75 days, To test whether the differences in rejection of thymus donor type skin were a property of the donor strain and also to test whether TL type might be involved C57-nunu recipients were used. These were not as well back-crossed as the BALB/c-nunu recipients but of 10 mice tested for TL type after thymus grafting eight were TL- like C57BL/6 (4). [When the thymus graft has been repopulated by host cells the TL antigen is of host type (1, 2)]. In #this laboratory the C57BL/6-nunu were very poorly viable, however, it was possible to keep some mice for long enough to make skin grafts and the results are given in Table 4. Two points can be made: firstly that AKR first grafts survive better than CBA, the reverse of the finding in BALB/c-nunu hosts and secondly a difference between the host and donor in TL type is not responsible for the unusual pattern of rejection.
RESPONSE
OF GRAFTEJ,
h’T_TIIES
XKR 1 2 3 1 5 6
TO
graft
LO 20 + 35 20 20 3.1
l)ONOK
103
:\STI(;J
~I~~.\'oI.oGY
Lymphocytes
25, 189-196 (1976)
Which
Differentiate
I. Response to MLC Determinants
in an Allogeneic
and Skin Grafts
from the Thymus
Thymus Donor Strain
B. KINDKEI) Basrl Imtitu
tc for Imntunology,
Grewacherstrassc Rcceiz’cd Fcbrrlaq
387,Postfach, 400. Rasel 5, Szvit~c~rlartd 18, 1976
INTKODGCTION When a nude mouse is grafted with a neonatal allogeneic thymus, host stem cells migrate into the graft and proliferate. From ahout 25 days after grafting it becomes difficult to find donor type lymphocytes in the graft (l-3) although a population of donor type lymphocytes does appear to persist and is more apparent if mice are examined some months after thymus grafting (4). \\‘hile the change from donor to host type cells is taking place in the thymus, cells also lcave tile and repopulate the peripheral lymphoid organs (1, 2, 5). Some donor tyl)e thymus T cells reach the periphery but the majority of the cells are of host origin (1 ). Tile immune responses in these animals are poorer than in normal mice but are comparable to those of nude mice with congenic thymus grafts (6, 7). These mice provide excellent material for studying the effects of the thymus on lymphocyte differentiation and particularly on the role of the thymus in recognition of self and non-self. From a theory proposed 1~. -Terne (a), one would predict that cells which differentiate in an allogeneic thymus cvould fail to react against tissues of the thymus donor strain. Pritchard and Rlicklem (3) found that this was the case for skin grafts but Seeger et al., (9) report the opposite for heart grafts and Kindred and Loor (6, 7) showed that skin grafts are sometimes accepted and sometimes rejected. The work presented here is a more thorough investigation of this problem and suggests that the animals are indeed tolerant to histocompatibilit~ antigens of the thymus donor strain but may respond to other, perhaps tissue specific, antigens. MATERIALS
AND r\lETHODS
11~01tscstvaim The strains used were BALB/cAnNIcrKb, C57BL/GNIcrKb, C&4/J, AKK/J, 129/J, A/J BALB/c-nunu and CS/“BL/G- 11ullu purchased from Gl. BomholtgSrd and a BALE/c-nunu stock made by direct backcrossing to BALB/cAnNIcrKb. These last were used only to establish that BALB/c-nunu grafted with a tllymus from the same sub-strain did not reject skin from that substrain. The A-TL- strain was derived from a cross-over (Boyse ef al., IO). They were kindly given to us by Dr. Donald Shreffler. 189 Copyright All rights
0 1976 by Academic Press, of reproduction in any form
190 Mixed Lymphocyte
B.
KINDRED
Culture
Lymph node cells were prepared under sterile conditions in Falcon plastic tubes, washed twice and diluted to 107/ml. Target cells were irradiated at 3300 r and 106 responders + lo6 target cells were incubated in each well of a microtiter plate. The medium used was RPM1 1640 medium (Microbiological Associates Inc., Bethesda, Md.), 10% fetal calf serum (Gibco) 0.03 A4 Hepes, and 2 mM glutamin, 100 III/ml of penicillin-streptomycin (Flow Laboratories Ltd., Irvine, Scotland). After 72 hr culture [“HI thymidine was added (2 &i/culture) and the cells were kept in culture for a further 24 hr then harvested, collected on Millipore filters and incubated overnight at 37°C in scintillation vials with 0.5 ml Protosol. The incorporated radioactivity was then measured. Skin Grafting Full thickness skin grafts were made as described by Ballantyne and Converse ( 11). Plasters were removed after 12 days and the grafts were examined daily for 2 weeks after which they were examined twice weekly. Histology Thymuses for histology were fixed in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. The preparation and examination was kindly performed by Dr. J$rgen Rygaard. RESULTS In all the experiments presented here the nude mice were left for at least 5 weeks after thymus grafting to allow ample time for host ‘type precursor cells to enter the thymus, differentiate and repopulate the peripheral lymphoid organs. Mixed Lymphocyte Reaction The ability of cells from BALB/c-nunu with BALB/c (H-2d) or allogeneic thymus grafts to react to the MLR determinants of the major histocompatibility complex was tested. The results are shown in Table 1. Mice with congenic, BALB/c, thymus grafts react equally strongly to C57BL/6J (H-2b) or AKR ( H-2k) and somewhat less strongly to CBA(H-2k). However, in general, mice with allogeneic thymus grafts react very poorly, if at all, to the donor strain or to strains having the same H-2 complex. The low responses may be real but they might also be due to “back stimulation” by the irradiated target cell,s as found by von Boehmer (12). There is one striking exception. The last of the mice listed with an AKR thymus graft reacted strongly to AKR cells. All the mice which produced a stimulation of five times or better against some target have been included in Table 1. However, these were by no means all the animals tested. About half the mice showed very feeble stimulation to all the targets. In some cases the background stimulation incorporation was very high and little increase was possible but this was not always so. There was no apparent correlation with age, status of skin grafts, presence of thymus graft or any other variable we could think of.
RESPONSE
OF GRAFTED
NUDES
TABLE Stimulation Grafts Given Incorporation Thymes
in Mixed Lymphocyte Cultures as [zH]thymidine Incorporation by Responder Cells + Irradiated
TO
I)OSOH
1
by Cells from BALB/c-nrmu Bearing Thymr~s by Responder Cells + Irradiated Target Cells: Responder Cells Target
graft AKR (H-2k)
CBA4(H-2k)
C57BL/6(H-2”)
3.7 6.0 5.2 4.2
5.6 7.6 7.6 11.0
5.80 6.W’ 3.8 9.7 6.1
5.1 10.0 2.4
2.4 1.8 1.0 2.2 0.9
CBA4
4.7
2.-t
11.3
AKR
2.8 2.4 21.3c
2.1 1.6 0.0
11.5 6.6 9.5 ___~~--
BALB,‘c
C57BL/6
101
ANTIGEXS
5.9 8.4 9.7 11.0
a This animal had accepted a C57BL/6 skin graft. h This animal had rejected a C57BL/6 skin graft. Tested in the same experiment c See text for further comment.
129(11-Pj
I .o
as ‘l.
The results of skin grafting experiments (Table 2) are more difficult to interpret than those of mixed lymphocyte cultures. Mice with congenic thymus grafts reject skin grafts from other strains. The rejection is slower than is found with normal mice but grafits are always rejected and second grafts are rejected more rapidly. This is also the case in recipients of allogeneic thymus grafts if they are grafted with skin from a third strain but the pattern of rejection of skin grafts from the thymus donor strain is very unusual. With C57BL/6, CBA or 129 donors half or more of the first ‘skin grafts are rejected but when animals which have rejected grafts are regrafted the second grafts are accepted. However, mice bearing AKR thymus grafts rarely accepted AKR skin grafts even if grafted two or three times. It is an interesting point that acceptance or rejection of skin grafts had no apparent effect on the behaviour of lymphocytes in mixed lymphocyte reaction e.g. the first and second C57BL/6 recipients listed in Table 1 were tested in MLC in the same experiment. Both had been grafted with C57BL/G skin; the first accepted the skin graft and the second rejected it but their reactions in MLC were very similar. As it has not been demonstrated that all donor type thymus cells are lost from a grafted thymus, it seemed possible that the differences in rejection patterns hetweerl CBA and C57BL/6J on the one hand and AKK on the other might be clue to differences in survival of a small but important residual population of donor thymus cells. To test this, BALB/c-nunu were grafted with AKR x C57BL/6 F, thymuses so that any residual population of donor thymocytes would bear both C57BL/6 and AKK histocompatibility antigens. Six such mice were given both C57BL/6
192
B.
KINDRED
TABLE Rejection
Thymus
graft
Skin graft
Rejection 10-15
BALB/c
C57BL/6
time in days
15-20
20-25 2
C57BL/6(lst) C57BL/6(2nd) CBA (1st) CBA (2nd) BALB/c
5 7 4 3
2
C57BL/6(lst) C57BL/6(2nd)b CBA (1st) CBA (2nd)
16
7
6
3 5
2
5
to
Not rejected” (survival 25 + days)
25+ 3
0
1 3(l) 14(6)
869
C57BL/6 (1st) C57BL/6(2nd) CBA (1st) CBA (2nd) AKR (1st) AKR (2nd)b AKR (3rd)” )
1 1 1 2 3 3 1
CBA
CBA CBA
(1st) (2nd) b
4
129
129 129
(1st) (2nd) b
1
AKR
2
Times for Skin Grafts from Various Strains BALB/c-nunu Bearing Thymus Grafts
1 1
1 2
1
2 3
4 1
2
l(l) l(l)
1 5 (2) 4(2) 1
1
1
3(2) 3
a The number in brackets is the number surviving with intact graft for more than 60 days. b 2nd and 3rd grafts were on mice which had rejected the 1st graft.
and AKR sskin grafts side by side. The rejection times for these grafts are shown in Table 3. The AKR grafts were all rejected but about half of the C57BL/6 grafts were accepted. Therefore differences in survival of C57BL/6 and AKR thymocytes cannot be responsible for the differences in pattern of skin graft rejection. Although nudes bearing an F1 thymus reject AKR skin they do not normally reject F1 skin. Six nudes were grafted with AKR x C57BL/6 ,thymuses and later with AKR x C57BL/6 skin. Only one of the skin grafts was rejected after 17 days. The survival times of the other mice with intact grafts were 21, 34, 44, 75 and 75 days, To test whether the differences in rejection of thymus donor type skin were a property of the donor strain and also to test whether TL type might be involved C57-nunu recipients were used. These were not as well back-crossed as the BALB/c-nunu recipients but of 10 mice tested for TL type after thymus grafting eight were TL- like C57BL/6 (4). [When the thymus graft has been repopulated by host cells the TL antigen is of host type (1, 2)]. In #this laboratory the C57BL/6-nunu were very poorly viable, however, it was possible to keep some mice for long enough to make skin grafts and the results are given in Table 4. Two points can be made: firstly that AKR first grafts survive better than CBA, the reverse of the finding in BALB/c-nunu hosts and secondly a difference between the host and donor in TL type is not responsible for the unusual pattern of rejection.
RESPONSE
OF GRAFTEJ,
h’T_TIIES
XKR 1 2 3 1 5 6
TO
graft
LO 20 + 35 20 20 3.1
l)ONOK
103
:\STI(;J
