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Comparison of the sensitivity of in vivo detection of immunological tolerance in the xenogeneic system
Immunology Letters, 4 (1982) 275-277 Elsevier Biomedical Press
C O M P A R I S O N O F T H E S E N S I T I V I T Y O F IN V I V O D E T E C T I O N O F I M M U N O L O G I C A L T O L E R A N C E IN T H E X E N O G E N E I C S Y S T E M Jitka CHUTNA, Milan HAgEK, Zden~k LODIN and Vladimfr HOL~drq Institute of Moleeular Genetics and Institute of Physiology, Czechoslovak Academy of Sciences, 166 37Prague 6, Czechoslovakia (Received 29 January 1982) (Accepted 12 February 1982)
1. Summary Three systems for the detection of xenogeneic tolerance in vivo are compared; skin xenografts, implantation of a tumour xenograft subcutaneously, and implantation ofa tumour xenograft into the brain. Implantation of tumour tissues into the brain has appeared to be the most sensitive test of immunological tolerance owing to the anatomical and immunological peculiarities of the brain.
2. Introduction
Different degrees of immunological tolerance may be found, depending on the detection system used. One detection system may detect the tolerance state in experimental animals and another fails to disclose it [1]. In our previous experiments [2} the in vivo and in vitro detection systems have been compared in the xenogeneic mouse-rat system, and an in vitro estimation of tolerance at the cellular level and the testing of humoral antibodies have been found more sensitive than skin graft testing. In thi.s paper we also compare the sensitivity of various in vivo tests in the xenogeneic mouse-rat system. It has appeared that the skin grafts, which are probably the most sensitive tests of immunity, are not a sensitive test of tolerance, whereas the tumour implants, particularly the tumour implants in the brain, may detect even a very low degree of immunological tolerance. Key words: in vivo tests -- skin grafts - turnout cells implantation into brain
0165-2478/82/0000-0000/$2.75 © Elsevier Biomedical Press
3. Materials and methods 3.1. Induction o f tolerance to xenogeneic cells Tolerance was induced in newborn (up to 20 h after birth) AVN rats by the intravenous injection of 43 X 106 bone marrow cells from C57BL/10Sn (hereafter B10) mice. Mouse bone marrow ceils depleted of T-cells by incubation with anti-Thy 1.2 serum and complement were used in 3 experiments and whole bone marrow cells in 2 experiments. At 8 weeks, all neonatally treated rats and control rats received B10 mouse skin grafts or tumour cell implants. 5 × 1 0 6 o r 20 × 106 tumour cells in a volume of 0.4 ml of phosphate-buffered saline (PBS) were injected subcutaneously, and 5 × 106 turnout cells in 0.05 ml of PBS were injected into the brain, in the occipital lobe in the right hemisphere 1.5-2 mm in depth [3]. 3.2. Characteristics o f tumour cells and an evaluation o f tumour growth Turnout cells were obtained from MC11 sarcoma which was induced by methylcholanthrene in B10 male mice. Cells were multiplied in tissue culture in Eagle's medium supplemented with 10% calf serum. Single cell suspensions were prepared by short trypsinization of cultured cells. After turnout cell implantation subcutaneously, the growth of turnouts was followed by palpation and estimated by twice weekly measurements of two perpendicular diameters of the tumour. After tumour cell inoculation into the brain, the animals were inspected daily and the effect of tumour growth was evaluated by death of the animals [3]. 275
Later it was found, however, that the whole bone marrow cells also did not induce a GVH reaction and that the same degree of tolerance was obtained in the model o f immunological tolerance used. In the first experiment, AVN rats were injected at birth with B10 mouse bone marrow cells depleted of T-cells or with whole bone marrow cells and tested with skin grafts from B10 mice at 8 weeks after birth. The differences in survival of skin xenografts between experimental and control groups were statistically significant. However, skin graft survival in groups of the neonatally treated animals was prolonged only slightly and all grafts were rejected within 13 days of grafting. In the control group, the grafts were rejected within 9 days. In a second experiment, the neonatally treated and control rats were given mouse MC11 tumour cells subcutaneously at 8 weeks after birth. One group received 5 × 106 tumour cells and the other group 20 × 106 tumour cells. No tumour growth was observed in the experimental and control groups after a smaller dose of 5 × 106 xenogeneic tumour cells (Fig. 1). In
3.3. Elimination o f T-cells from BI O mouse bone marrow cell suspension Monoclonal anti-Thy 1.2 antibodies were used to eliminate T-cells. B10 mouse bone marrow cells at a concentration of 50 × 10 6 cells/ml o f antibodies diluted 1:100 were incubated for 30 rain at room temperature and then for 30 min at 37°C with an equal volume of rabbit serum (diluted 1:4) which had been preabsorbed with mouse cells and served as a source of complement. Cells were washed twice before inoculation into animals. 3.4. Statistical evaluation The significance of the differences between experimental and control groups was determined using the test of the difference between two binomial variables and a test described by Sid~ik and Vondr~i~ek [4].
4. Results Xenogeneic bone marrow cells, which had been depleted ofT-cells to prevent a graft-versus-host (GVH) reaction, were at first used for tolerance induction,
H
Controls - 2 0 x 4 0 6 t u m o u r cells
~ - - . - A Controls - 5w4OSturnour cells c)----o Neonatally treated - 20x4Or°turnour cells z~. . . . z~ Neonatally treoted - 5xtOStumour cells
200 g-
E E is0
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35 25 30 Doy.S Qf~er i~plontotio~ of turnout cells 20
~N
~0
Fig. 1. Tumour growth following inoculation of mouse tumour cells subcutaneously into treated and control rats. The curve depicts the average turnout size in individual experimental and control groups at various time intervals after implantation. Experimental groups received at birth an intravenous injection of 43 X 106 xenogeneic bone marrow cells depeleted of T-cells. 276
Table 1 Mortality of control and neonatally treated rats following inoculation of 5 × 106 xenogeneic tumour cells into the brain Animalsa
Mortality of rats
Deaths (days)
Neonatally treated I Neonatally treated II Controls untreated
12/12b 7/8 3/11
6-8 10-24 7
aNeonatally treated 1 rats received B 10 mouse bone marrow cells depleted of T-cells by incubation with anti-Thy 1.2 serum and complement. Neonatally treated II rats were given whole bone marrow cells from B10 mice. bNumerator, number of dead animals; denominator, total number of animals 15 control rats inoculated with 20 X 106 tumour cells, an initial small tumour growth was noted, but all tumours regressed and disappeared within 14 days. In the group consisting of 10 experimental rats inoculated at birth with xenogeneic bone marrow cells, the turnouts grew in 5 animals for longer periods than in the controls and were still detectable on days 1 5 - 1 9 . In one rat, the turnout grew progressively and was rejected as late as day 40. In a third experiment a great part of the experimental rats treated at birth with xenogeneic bone marrow cells died after implantation o f 5 X 106 xenogeneic tumour cells into the brain (Table 1). M1 of the 12 experimental animals died in the group of rats injected at birth with B10 mouse bone marrow cells depleted of T-cells. Seven of 8 rats neonatally inoculated with whole bone marrow cells succumbed. The neonatally treated animals died within 6 - 2 4 days of implantation of xenogeneic tumour cells. Some control rats, like the controls in the allogeneic system, died after implantation of 5 X 106 xenogeneic tumour cells, but the difference between the control group (3 of 11 rats died) and the experimental group (19 of 20 rats succumbed) were statistically highly significant (P <0.001 ).
5. Discussion In our experiments on rats, no marked degree of tolerance to xenogeneic skin grafts and no manifestations of a GVH reaction were seen following an intravenous injection of xenogeneic bone marrow cells at
birth. This suggested that there was a minimum of complete xenogeneic tolerance. However. the turnout xenografts detected a given degree of tolerance more sensitively and the tumours grew much better in animals treated at birth with xenogeneic bone marrow cells than in the controls. Implantation of a tumour into the brain appeared to be the most sensitive test of this low degree of tolerance because the "tolerant" and control animals markedly differed in mortality. The difference in the sensitivity of an in vivo detection of immunological tolerance may be due to the brain milieu where an inoculum is administered and to the known ability of the tumours to grow against immunity, and perhaps to the peculiarities of transplantation immunity in the brain [3,5]. From the data reported here it can be concluded that among the hitherto known detection systems implantation of turnout tissues into the brain is the most sensitive test for revealing the lowest degree of immunological tolerance. Also pertinent here is the fact that the tumour growing in the brain kills the animal before effective immune responses develop. Therefore even a slighter weakening of the immunity system may be detected. Thus, less sensitive tests of immunity appear to be more sensitive tests of immunological tolerance.
Acknowledgements We thank Dr. J. Bubenl"k of this Institute for providing MC 11 tumour cells, Dr. L. Steiner, Department of Biology, Massachusetts Institute o f Technology, Cambridge, MA, U.S.A. for generously supplying monoclonal anti-Thy 1.2 antibodies, and Dr. V. Matougek of this Institute for performing statistical analysis.
References [1] Ha~ek, M., Holfifi, V. and Kousalovfi,M. (1981) Immunol. Lett. 3, 183-186. [2] Chutn~, J., Holfifi,V. and Ha~ek,M. (1981) Cell Immunol. 63,193-197. [3] Ha~ek, M., Chutnfi, J., Shlde~ek, M. and Lodin, Z. (1977) Nature (London) 268, 68-69. [4] Sidfik, Z., Vondr~ek, J. (1957) Apl. matem. (in Czech) 2, 215-221. [ 5] Barker, C. F. and Billingham, R. E. (1977) Adv. Immunol. 25, 1-49. 277
C O M P A R I S O N O F T H E S E N S I T I V I T Y O F IN V I V O D E T E C T I O N O F I M M U N O L O G I C A L T O L E R A N C E IN T H E X E N O G E N E I C S Y S T E M Jitka CHUTNA, Milan HAgEK, Zden~k LODIN and Vladimfr HOL~drq Institute of Moleeular Genetics and Institute of Physiology, Czechoslovak Academy of Sciences, 166 37Prague 6, Czechoslovakia (Received 29 January 1982) (Accepted 12 February 1982)
1. Summary Three systems for the detection of xenogeneic tolerance in vivo are compared; skin xenografts, implantation of a tumour xenograft subcutaneously, and implantation ofa tumour xenograft into the brain. Implantation of tumour tissues into the brain has appeared to be the most sensitive test of immunological tolerance owing to the anatomical and immunological peculiarities of the brain.
2. Introduction
Different degrees of immunological tolerance may be found, depending on the detection system used. One detection system may detect the tolerance state in experimental animals and another fails to disclose it [1]. In our previous experiments [2} the in vivo and in vitro detection systems have been compared in the xenogeneic mouse-rat system, and an in vitro estimation of tolerance at the cellular level and the testing of humoral antibodies have been found more sensitive than skin graft testing. In thi.s paper we also compare the sensitivity of various in vivo tests in the xenogeneic mouse-rat system. It has appeared that the skin grafts, which are probably the most sensitive tests of immunity, are not a sensitive test of tolerance, whereas the tumour implants, particularly the tumour implants in the brain, may detect even a very low degree of immunological tolerance. Key words: in vivo tests -- skin grafts - turnout cells implantation into brain
0165-2478/82/0000-0000/$2.75 © Elsevier Biomedical Press
3. Materials and methods 3.1. Induction o f tolerance to xenogeneic cells Tolerance was induced in newborn (up to 20 h after birth) AVN rats by the intravenous injection of 43 X 106 bone marrow cells from C57BL/10Sn (hereafter B10) mice. Mouse bone marrow ceils depleted of T-cells by incubation with anti-Thy 1.2 serum and complement were used in 3 experiments and whole bone marrow cells in 2 experiments. At 8 weeks, all neonatally treated rats and control rats received B10 mouse skin grafts or tumour cell implants. 5 × 1 0 6 o r 20 × 106 tumour cells in a volume of 0.4 ml of phosphate-buffered saline (PBS) were injected subcutaneously, and 5 × 106 turnout cells in 0.05 ml of PBS were injected into the brain, in the occipital lobe in the right hemisphere 1.5-2 mm in depth [3]. 3.2. Characteristics o f tumour cells and an evaluation o f tumour growth Turnout cells were obtained from MC11 sarcoma which was induced by methylcholanthrene in B10 male mice. Cells were multiplied in tissue culture in Eagle's medium supplemented with 10% calf serum. Single cell suspensions were prepared by short trypsinization of cultured cells. After turnout cell implantation subcutaneously, the growth of turnouts was followed by palpation and estimated by twice weekly measurements of two perpendicular diameters of the tumour. After tumour cell inoculation into the brain, the animals were inspected daily and the effect of tumour growth was evaluated by death of the animals [3]. 275
Later it was found, however, that the whole bone marrow cells also did not induce a GVH reaction and that the same degree of tolerance was obtained in the model o f immunological tolerance used. In the first experiment, AVN rats were injected at birth with B10 mouse bone marrow cells depleted of T-cells or with whole bone marrow cells and tested with skin grafts from B10 mice at 8 weeks after birth. The differences in survival of skin xenografts between experimental and control groups were statistically significant. However, skin graft survival in groups of the neonatally treated animals was prolonged only slightly and all grafts were rejected within 13 days of grafting. In the control group, the grafts were rejected within 9 days. In a second experiment, the neonatally treated and control rats were given mouse MC11 tumour cells subcutaneously at 8 weeks after birth. One group received 5 × 106 tumour cells and the other group 20 × 106 tumour cells. No tumour growth was observed in the experimental and control groups after a smaller dose of 5 × 106 xenogeneic tumour cells (Fig. 1). In
3.3. Elimination o f T-cells from BI O mouse bone marrow cell suspension Monoclonal anti-Thy 1.2 antibodies were used to eliminate T-cells. B10 mouse bone marrow cells at a concentration of 50 × 10 6 cells/ml o f antibodies diluted 1:100 were incubated for 30 rain at room temperature and then for 30 min at 37°C with an equal volume of rabbit serum (diluted 1:4) which had been preabsorbed with mouse cells and served as a source of complement. Cells were washed twice before inoculation into animals. 3.4. Statistical evaluation The significance of the differences between experimental and control groups was determined using the test of the difference between two binomial variables and a test described by Sid~ik and Vondr~i~ek [4].
4. Results Xenogeneic bone marrow cells, which had been depleted ofT-cells to prevent a graft-versus-host (GVH) reaction, were at first used for tolerance induction,
H
Controls - 2 0 x 4 0 6 t u m o u r cells
~ - - . - A Controls - 5w4OSturnour cells c)----o Neonatally treated - 20x4Or°turnour cells z~. . . . z~ Neonatally treoted - 5xtOStumour cells
200 g-
E E is0
E
/
/o-----o-
. . . .
/
\ \
/
\
/
O
0
\
/
.w100
/
u3
/
\ \ \ \ \ \ \
SO
\ \
I
0
5
t0
tS
!
t
!
!
35 25 30 Doy.S Qf~er i~plontotio~ of turnout cells 20
~N
~0
Fig. 1. Tumour growth following inoculation of mouse tumour cells subcutaneously into treated and control rats. The curve depicts the average turnout size in individual experimental and control groups at various time intervals after implantation. Experimental groups received at birth an intravenous injection of 43 X 106 xenogeneic bone marrow cells depeleted of T-cells. 276
Table 1 Mortality of control and neonatally treated rats following inoculation of 5 × 106 xenogeneic tumour cells into the brain Animalsa
Mortality of rats
Deaths (days)
Neonatally treated I Neonatally treated II Controls untreated
12/12b 7/8 3/11
6-8 10-24 7
aNeonatally treated 1 rats received B 10 mouse bone marrow cells depleted of T-cells by incubation with anti-Thy 1.2 serum and complement. Neonatally treated II rats were given whole bone marrow cells from B10 mice. bNumerator, number of dead animals; denominator, total number of animals 15 control rats inoculated with 20 X 106 tumour cells, an initial small tumour growth was noted, but all tumours regressed and disappeared within 14 days. In the group consisting of 10 experimental rats inoculated at birth with xenogeneic bone marrow cells, the turnouts grew in 5 animals for longer periods than in the controls and were still detectable on days 1 5 - 1 9 . In one rat, the turnout grew progressively and was rejected as late as day 40. In a third experiment a great part of the experimental rats treated at birth with xenogeneic bone marrow cells died after implantation o f 5 X 106 xenogeneic tumour cells into the brain (Table 1). M1 of the 12 experimental animals died in the group of rats injected at birth with B10 mouse bone marrow cells depleted of T-cells. Seven of 8 rats neonatally inoculated with whole bone marrow cells succumbed. The neonatally treated animals died within 6 - 2 4 days of implantation of xenogeneic tumour cells. Some control rats, like the controls in the allogeneic system, died after implantation of 5 X 106 xenogeneic tumour cells, but the difference between the control group (3 of 11 rats died) and the experimental group (19 of 20 rats succumbed) were statistically highly significant (P <0.001 ).
5. Discussion In our experiments on rats, no marked degree of tolerance to xenogeneic skin grafts and no manifestations of a GVH reaction were seen following an intravenous injection of xenogeneic bone marrow cells at
birth. This suggested that there was a minimum of complete xenogeneic tolerance. However. the turnout xenografts detected a given degree of tolerance more sensitively and the tumours grew much better in animals treated at birth with xenogeneic bone marrow cells than in the controls. Implantation of a tumour into the brain appeared to be the most sensitive test of this low degree of tolerance because the "tolerant" and control animals markedly differed in mortality. The difference in the sensitivity of an in vivo detection of immunological tolerance may be due to the brain milieu where an inoculum is administered and to the known ability of the tumours to grow against immunity, and perhaps to the peculiarities of transplantation immunity in the brain [3,5]. From the data reported here it can be concluded that among the hitherto known detection systems implantation of turnout tissues into the brain is the most sensitive test for revealing the lowest degree of immunological tolerance. Also pertinent here is the fact that the tumour growing in the brain kills the animal before effective immune responses develop. Therefore even a slighter weakening of the immunity system may be detected. Thus, less sensitive tests of immunity appear to be more sensitive tests of immunological tolerance.
Acknowledgements We thank Dr. J. Bubenl"k of this Institute for providing MC 11 tumour cells, Dr. L. Steiner, Department of Biology, Massachusetts Institute o f Technology, Cambridge, MA, U.S.A. for generously supplying monoclonal anti-Thy 1.2 antibodies, and Dr. V. Matougek of this Institute for performing statistical analysis.
References [1] Ha~ek, M., Holfifi, V. and Kousalovfi,M. (1981) Immunol. Lett. 3, 183-186. [2] Chutn~, J., Holfifi,V. and Ha~ek,M. (1981) Cell Immunol. 63,193-197. [3] Ha~ek, M., Chutnfi, J., Shlde~ek, M. and Lodin, Z. (1977) Nature (London) 268, 68-69. [4] Sidfik, Z., Vondr~ek, J. (1957) Apl. matem. (in Czech) 2, 215-221. [ 5] Barker, C. F. and Billingham, R. E. (1977) Adv. Immunol. 25, 1-49. 277