- Email: [email protected]
Evaluation of microchimerism after orthotopic liver transplantation between allogeneic mice
ELSEVIER
Evaluation of Microchimerism Between Allogeneic Mice A. Sugioka,
After Orthotopic
Liver Transplantation
M. Morita, T. Esaki, A. Hasumi, and Y. Kurosawa
I
MMUNOLOGIC phenomena, such as the ability to discriminate between self and nonself, are believed to be responsible for the rapid rejection of organ grafts between allogeneic individuals in the absence of immunosuppressive agents. The highly polymorphic molecules of the major histocompatibility complex (MHC) appear to act as barriers in such transplantation. However, while these considerations are absolutely valid in the case of bone marrow and skin transplantation, liver transplantation in domestic pigs and in rodents seems to provide exceptions to this rule.‘.* Liver grafts between some combinations of allogeneic rats and between many combinations of allogeneic mice are accepted without immunosuppressive interMoreover, liver grafts of this type seem to ventions.” induce donor-specific immunotolerance in the recipient animals.4 Even in humans, it has been shown that unresponsiveness to the grafts has been achieved in some patients with long-term successful engraftment.’ With respect to the mechanism of graft acceptance and the induction of tolerance, two different hypotheses have been proposed. Microchimerism that results from the migration of passenger leukocytes, originally present in the grafted liver, could form the basis for induction of tolerance.’ Both dendritic and hematopoietic stem cells have been proposed to be responsible for such a phenomenon.‘-’ Alternatively, soluble MHC class I molecules, presumably secreted from the parenchymal cells in the liver grafts, could block the binding of alloantibodies and of cytotoxic T cells directed toward the allografts, thereby inducing the unresponsiveness.‘OX” It was reported recently that soluble HLA class I molecules induced apoptosis in alloreactive cytotoxic T lymphocytes.” In the present study, we established an orthotopic liver transplantation system using mice (OMLT) according to the previously published procedure with minor modifications.” We searched for evidence of the presence of cells that expressed the donor-type MHC class I and/or class II gene. Since the MHC loci of the mouse (H-2) have been precisely analyzed and, moreover, since many lines of H-2 congenic mice have been established, we were able to investigate possible microchimerism in the liver-grafted mice using reverse transcriptase-polymerase chain reaction (RT-PCR). 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas,
New York, NY 10010
MATERIALS
AND
METHODS
Male 10 to 12-week-old mice from H-2 congenic strains derived from mice with the C57BUlO background were used as follows: BlO.BR/SgSn (K’I-AkI-EkDk) and BlO.D2/SgSn (KdI-AdI-EdDd). Eleven transplants (OMLT) were performed from BlO.DZ into BlO.BR mice (n = 2) and from BlO.BR into BlO.D2 mice (n = 9). OMLT was performed by the cuff technique.14 The suprahepatic inferior vena cava was anastomosed by suturing, and the portal vein and infrahepatic vena cava were reconstructed with the cuff technique, as in the transplantation of rat liver that was described by Ramada and Calne. I4 Furthermore, the bile duct was reconstructed with a single stent tube. Animals were sacrificed on days 7 (n = 3), 14 (n = 3) 21 (n = 2). 28 (n = l), 35 (n = l), and 63 (n = 1) after OMLT. Total RNA was extracted from the spleen, thymus, blood, and kidney. RT-PCR was performed as follows. cDNA was synthesized by reverse transcriptase using 2 pg of total RNA as a template and oligo dT as a primer. Four sets of primers were synthesized for specific amplification of transcripts that encoded H-2Kd, H-2K”, I-Ad. and I-Ak, respectively. The sequences of primers were designed on the basis of published sequences”,r6 and are shown in Table 1. The specificity of the amplified fragments was confirmed by digestion with restriction enzymes whose sites included the polymorphic residues in each fragment. The expected sizes of the products are summarized in Table 1. The conditions for PCR were essentially the same as those described elsewhere.” PCR was performed for 30 cycles and the products were separated on a 2% agarose gel and visualized by staining with ethidium bromide. RESULTS Orthotopic
Liver Transplantation
Between
Allogeneic
Mice
Between August 1994 and July 1996, we performed OMLT a total of 700 times, finally achieving a l-week survival rate of 97% as a result of improvements in our technique. OMLT was performed with 32 allogeneic combinations using 13 inbred strains (the details will be published elseFrom the Department of Surgery and Institute for Comprehensive Medical Science, Fujita Health University School of Medicine, Aichi, Japan. Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (grant 0745723) Address reprint requests to Atsushi Sugioka, MD, Department of Surgery, Fujita Health University School of Medicine, Toyoake, Aichi 470-l 1, Japan.
0041-l 345/97/$17.00 PII SO041 -1345(96)00539-8
1189
Transplantation
Proceedings,
29, 1189-l 192 (1997)
SUGIOKA,
1190
MORITA, ESAKI ET AL
Table 1. Primers for Specific Amplification by RT-PCR of the Transcripts That Encoded H-2Kd, H-2Kk, I-Ad, and I-Ak Diaestion With Restriction Enzyme Sequence of Primers
H-Z Haplotype
5’-GAGCAGACACAGAG 5’-AACTTGAGATCTGGG 5’-CGlTCCAACGGATGTAC 5’-ATC-ITCAGGTCTGCT 5’-TACGAGGGGCCGGAGACCAG 5’-GGTGTAGACCTCTCCCTGAT 5’-TGCACCAGTTCCAGCCC-IX 5’-GGTGTAGACCTCTCCCCGCC
Kd
Kk I-Ad I-A”
‘X2+4
Product Sizes (bp)
Product Size [bp)
Enzyme
408
Kpnl
252+4, 152+4
311
Bgll
179+3, 129+3
273
Hphl
150+1,122+1
497
Taql Hphl
398+2,97+2 360+1, 116+2, 19+1
indicates that the double-stranded region was 252 bp long with a four-nucleotide single-stranded portion.
where). A summary of our results is given here. In all the cases, liver grafts were accepted spontaneously without immunosuppression. Moreover, donor-specific skin grafts were accepted when skin transplantation was performed 60 days after liver transplantation. However, 30 days after liver transplantation, such skin grafts were rejected. Nevertheless, even when the skin was rejected, the grafted liver was retained in many cases. In some cases, the mice did die because of rejection of the liver. Thus, more than 30 days were necessary for the induction of complete immunotolerance. When skin transplantation was performed 7 days before liver transplantation, the grafted mouse died within 1 week after the liver transplant as a result of rejection of the liver. These results indicated that OMLT might provide
a. Specificity for class I (K) primersfor Kd t
primersfor Kk I
E. Sensitivity for class I (K)
a superior experimental model for studies designed to clarify the mechanism of graft acceptance and the induction of tolerance. Using one combination of H-2 allotypes, H-2d and H-2k, we examined microchimerism after liver transplantation in the present study. Analysis of Microchimerism
by RT-PCR
We prepared four sets of primers that we had designed for specific amplification of transcripts that encoded H-2Kk, Kd, I-Ak, and I-Ad, respectively, as indicated in Table 1. The specificity of the amplification of fragments was determined from the sizes of fragments, as indicated by their mobility on a gel. Specific amplification was observed, as shown in Fig la and lb. Digestion with restriction enzymes confirmed
b. Specificity for class II (IA) primersfor lAd I-
primersforIAk
d. Sensitivity for class II (IA)
Fig 1. Evaluation of the specificity (a, b) and sensitivity (c, d) of RT-PCR using tissues from untreated animals, namely, BlO.D2 (d-haplotype) and BlO.BR (k-haplotype). Highly specific bands at the expected position, representing transcripts of each class I and class II gene, were visualized after RT-PCR with four sets of primers. The sensitivity of RT-PCR was examined with mixtures of serially diluted samples of RNA and the limit of detection was between l/lo3 and 1/104.
MICROCHIMERISM
IN MICE
1191
P~~IIWS fix Kd prinws for KL
n-
-Kmhp ( Kd: donor ~ypc) 311 bp (K’: rccipicnt typ)
Detection of microchimerism after OMLT. A BlO.D2 (d-haplotype) graft was transplanted into a BlO.BR (k-haplotype) recipient. Fig 2. On day 14, total RNA was extracted from the spleen, thymus, and blood of the recipient. The results after RT-PCR revealed clear evidence of the transcription of both donor-type class I and class II genes, demonstrating the existence of microchimerism.
the amplification of the expected fragments (data not shown). We also examined the sensitivity of RT-PCR. Samples of RNA prepared from the liver of BlO.BR (k-haplotype) and BlO.D2 (d-haplotype) mice were mixed at ratios of 104:1 to 1:104 (w/w), and RT-PCR was performed with four sets of primers. The limit of detection was between l/10” and l/lo4 under the conditions used in this study, as shown in Fig lc and Id. The presence of transcripts that encoded donor-derived H-2K and I-A was examined in grafted mice on days 7, 14, 21,28,35, and 63. A typical example, revealing the presence of microchimerism, is shown in Fig 2. In one case, we found microchimerism of class II-expressing cells in the spleen and thymus on day 7. On day 14, all three cases examined showed microchimerism in the spleen, thymus, and blood. Moreover, in two cases, transcripts for both donor-derived class I and II molecules were detected, and in one case, only transcripts for class II molecules were detected. On day 21,
Table
2. Detection
split results were obtained. However, from day 28 to day 63, the donor-derived transcripts encoding either class I or II molecules were no longer detectable. These results are summarized in Table 2. As mentioned above, more than 30 days were required after liver transplantation for complete induction of immunotolerance to donor-type skin. Nevertheless, graft acceptance and induction of tolerance were consistently achieved regardless of the presence or absence of detectable microchimerism.
DISCUSSION
The system for OMLT has been well established in our laboratory. Successful OMLT can now be performed twice daily with a very high success rate. The results of OMLT with a large variety of allogeneic combinations of available laboratory strains indicate that spontaneous graft acceptance and induction of tolerance are general phenomena
of Microchimerism
by FIT-PCR After OMLT Microchimerism
Strain Combination Days After OMLT 7
14
21 28
Donor
Thymus
Spleen
I
Blood Class I
Class II
NT
NT NT
NT _
NT
NT NT
NT
_
_
_
_
+
NT
NT
Class I
Class II
Class I
D2
_
_
_
D2
_
+ _
NT _
NT
D2
+ _
Class
BR BR BR
Kidney Class II
Class II
Recipient
BR
D2
+
+
+
+
+
D2
BR
+
+
+
_
BR
D2
+ _
+
+ _
+
NT
NT
NT
NT
BR
D2
+
D2
+ _
+ _
NT _
NT _
+ _
+
BR
+ _
BR
D2
_
_
_
_
_
_
_
NT
NT
_
NT
NT
35
BR
D2
_
63
D2
BR
_
Abbreviation: NT, not tested.
_
_ _
_
_
1192
after OMLT. We recently used several strains of wild mice that have been established at the National Institute of Genetics at Mishima in Japan for OMLT. The results were essentially the same as those obtained with the laboratory strains and appear to be different from those obtained with rats. In the case of rat liver transplantation, three different types of results have been obtained, depending on the combination of strains.18 In one group of rats, for example, the combination of DA(RTla) as donor and PVG(RT1’) as recipient, permanent acceptance and induction of tolerance were observed consistently, as in the present study with mice. In another group of rats, for example, with DA(RTla) as donor and LEW(RTll) as recipient, the grafted rats died because of rejection about 11 days after the operation. In the third group, for example, with BN(RTlm) as donor and LEW(RT1’) as recipient, the grafted rats survived for a prolonged period but ultimately died. We confirmed these results by 600 operations for transplantation of rat liver. As long as the operation was carried out under the appropriate conditions, the above-described results were obtained consistently. Thus, the acceptance or rejection by rats of grafted livers appears to be genetically determined. The phenomenon of microchimerism after solid-organ transplantation has been well established.’ It remains unclear, however, whether the microchimerism per se is the cause of induction of tolerance or a consequence. Moreover, the detection of donor cell-derived genomic DNA by PCR that has usually been utilized to demonstrate the presence of microchimerism is too sensitive for suitable evaluation and, furthermore, such detection is not quantitative. In the present study, we used RT-PCR. Our results indicated that the number of the donor-derived MHC class II-expressing cells reached a maximum 2 weeks after liver transplantation and then decreased. Since the sensitivity of RT-PCR was between l/lo3 and 1/104, we cannot ignore the possibility that donor-derived cells remained viable for a long time. However, we can draw the following conclusions. (1) The donor-derived cells migrated throughout the recipient’s body after the liver transplantation, with microchimerism developing in many cases and possibly even in all cases. MHC class II-expressing cells that might have had antigen-presenting capability were included among the migrating cells. (2) The numbers of donor-derived cells increased 2 and 3 weeks after the operation. However, this increase in numbers of migrating cells did not seem to be essential for the induction of tolerance since the extent of the increase appeared to be variable even though the tolerance was induced in all cases. (3) Four weeks after the
SUGIOKA,
MORITA, ESAKI ET AL
operation, microchimerism, even if it remained, fell below the limit of detection by RT-PCR. (4) More than 30 days were required for the full induction of tolerance. (5) About 30 days after liver transplantation, donor-specific tolerance appeared to have been only partly induced, being related perhaps to the phenomenon known as “split tolerance.“4 (6) After prior sensitization with a skin graft, a liver graft never induced tolerance. Pluripotent hematopoietic stem cells reside in the liver.8s9 The transient appearance of donor-derived cells between 1 and 3 weeks after liver transplantation might have been due to the presence of such cells. It has been argued that the number of donor-derived cells is crucial in the deletion of donor-reactive T cells and skin graft acceptance and that there exists a threshold with respect to the extent of chimerism that is required for the induction of tolerance.19”’ Our results suggest that, although microchimerism was clearly detected, it was a transient phenomenon. Four weeks after operation when tolerance had been partially induced, the extent of microchimerism fell below the detectable level. Thus, it seems difficult to explain the induction of tolerance solely in terms of the existence of microchimerism. REFERENCES
1. Calne RY, et al: Nature 223:472, 1969
2. 3. 4. 5. 6. 7.
Kamada N, Davies HFFS, Roser B: Nature 292:840, 1981 Qian S, et al: Hepatology 19:916, 1994 Dahmen U, et al: Transplantation 58:1, 1994 Starzl TE, et al: Hepatology 17:1127, 1993 Woo J, et al: Transplantation 58:484, 1994
Lu L, et al: J Exp Med 182:379, 1995 8. Taniguchi H, et al: Nature Med 2:198, 1996 9. Murase N, et al: Transplantation 61:1, 1996 10. Buelow R, Burlingham WJ, Clayberger C: Transplantation 59:649, 1995 11. Sriwatanawongsa V, Davies HFFS, Calne RY: Nature Med 1:428, 1995 12. Zavazava N, Kronke M: Nature Med 2:1005, 1996 13. Qian S, et al: Transplantation 52:562, 1991 14. Kamada N, Caine RY: Transplantation 28:47, 1979 15. Kuhner MK, Goodenow RS: Immunogenetics 30:458, 1989 16. Choi E, et al: Science 221:283, 1983 17. Saiki RK, et al: Science 239:487, 1988 18. Kamada N: Experimental Liver Transplantation. Florida: CRC Press; 1988 19. Murase N, et al: Transplantation 60:158, 1995 20. Taniguchi H, et al: J Immunol 155:5631, 1995
Evaluation of Microchimerism Between Allogeneic Mice A. Sugioka,
After Orthotopic
Liver Transplantation
M. Morita, T. Esaki, A. Hasumi, and Y. Kurosawa
I
MMUNOLOGIC phenomena, such as the ability to discriminate between self and nonself, are believed to be responsible for the rapid rejection of organ grafts between allogeneic individuals in the absence of immunosuppressive agents. The highly polymorphic molecules of the major histocompatibility complex (MHC) appear to act as barriers in such transplantation. However, while these considerations are absolutely valid in the case of bone marrow and skin transplantation, liver transplantation in domestic pigs and in rodents seems to provide exceptions to this rule.‘.* Liver grafts between some combinations of allogeneic rats and between many combinations of allogeneic mice are accepted without immunosuppressive interMoreover, liver grafts of this type seem to ventions.” induce donor-specific immunotolerance in the recipient animals.4 Even in humans, it has been shown that unresponsiveness to the grafts has been achieved in some patients with long-term successful engraftment.’ With respect to the mechanism of graft acceptance and the induction of tolerance, two different hypotheses have been proposed. Microchimerism that results from the migration of passenger leukocytes, originally present in the grafted liver, could form the basis for induction of tolerance.’ Both dendritic and hematopoietic stem cells have been proposed to be responsible for such a phenomenon.‘-’ Alternatively, soluble MHC class I molecules, presumably secreted from the parenchymal cells in the liver grafts, could block the binding of alloantibodies and of cytotoxic T cells directed toward the allografts, thereby inducing the unresponsiveness.‘OX” It was reported recently that soluble HLA class I molecules induced apoptosis in alloreactive cytotoxic T lymphocytes.” In the present study, we established an orthotopic liver transplantation system using mice (OMLT) according to the previously published procedure with minor modifications.” We searched for evidence of the presence of cells that expressed the donor-type MHC class I and/or class II gene. Since the MHC loci of the mouse (H-2) have been precisely analyzed and, moreover, since many lines of H-2 congenic mice have been established, we were able to investigate possible microchimerism in the liver-grafted mice using reverse transcriptase-polymerase chain reaction (RT-PCR). 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas,
New York, NY 10010
MATERIALS
AND
METHODS
Male 10 to 12-week-old mice from H-2 congenic strains derived from mice with the C57BUlO background were used as follows: BlO.BR/SgSn (K’I-AkI-EkDk) and BlO.D2/SgSn (KdI-AdI-EdDd). Eleven transplants (OMLT) were performed from BlO.DZ into BlO.BR mice (n = 2) and from BlO.BR into BlO.D2 mice (n = 9). OMLT was performed by the cuff technique.14 The suprahepatic inferior vena cava was anastomosed by suturing, and the portal vein and infrahepatic vena cava were reconstructed with the cuff technique, as in the transplantation of rat liver that was described by Ramada and Calne. I4 Furthermore, the bile duct was reconstructed with a single stent tube. Animals were sacrificed on days 7 (n = 3), 14 (n = 3) 21 (n = 2). 28 (n = l), 35 (n = l), and 63 (n = 1) after OMLT. Total RNA was extracted from the spleen, thymus, blood, and kidney. RT-PCR was performed as follows. cDNA was synthesized by reverse transcriptase using 2 pg of total RNA as a template and oligo dT as a primer. Four sets of primers were synthesized for specific amplification of transcripts that encoded H-2Kd, H-2K”, I-Ad. and I-Ak, respectively. The sequences of primers were designed on the basis of published sequences”,r6 and are shown in Table 1. The specificity of the amplified fragments was confirmed by digestion with restriction enzymes whose sites included the polymorphic residues in each fragment. The expected sizes of the products are summarized in Table 1. The conditions for PCR were essentially the same as those described elsewhere.” PCR was performed for 30 cycles and the products were separated on a 2% agarose gel and visualized by staining with ethidium bromide. RESULTS Orthotopic
Liver Transplantation
Between
Allogeneic
Mice
Between August 1994 and July 1996, we performed OMLT a total of 700 times, finally achieving a l-week survival rate of 97% as a result of improvements in our technique. OMLT was performed with 32 allogeneic combinations using 13 inbred strains (the details will be published elseFrom the Department of Surgery and Institute for Comprehensive Medical Science, Fujita Health University School of Medicine, Aichi, Japan. Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (grant 0745723) Address reprint requests to Atsushi Sugioka, MD, Department of Surgery, Fujita Health University School of Medicine, Toyoake, Aichi 470-l 1, Japan.
0041-l 345/97/$17.00 PII SO041 -1345(96)00539-8
1189
Transplantation
Proceedings,
29, 1189-l 192 (1997)
SUGIOKA,
1190
MORITA, ESAKI ET AL
Table 1. Primers for Specific Amplification by RT-PCR of the Transcripts That Encoded H-2Kd, H-2Kk, I-Ad, and I-Ak Diaestion With Restriction Enzyme Sequence of Primers
H-Z Haplotype
5’-GAGCAGACACAGAG 5’-AACTTGAGATCTGGG 5’-CGlTCCAACGGATGTAC 5’-ATC-ITCAGGTCTGCT 5’-TACGAGGGGCCGGAGACCAG 5’-GGTGTAGACCTCTCCCTGAT 5’-TGCACCAGTTCCAGCCC-IX 5’-GGTGTAGACCTCTCCCCGCC
Kd
Kk I-Ad I-A”
‘X2+4
Product Sizes (bp)
Product Size [bp)
Enzyme
408
Kpnl
252+4, 152+4
311
Bgll
179+3, 129+3
273
Hphl
150+1,122+1
497
Taql Hphl
398+2,97+2 360+1, 116+2, 19+1
indicates that the double-stranded region was 252 bp long with a four-nucleotide single-stranded portion.
where). A summary of our results is given here. In all the cases, liver grafts were accepted spontaneously without immunosuppression. Moreover, donor-specific skin grafts were accepted when skin transplantation was performed 60 days after liver transplantation. However, 30 days after liver transplantation, such skin grafts were rejected. Nevertheless, even when the skin was rejected, the grafted liver was retained in many cases. In some cases, the mice did die because of rejection of the liver. Thus, more than 30 days were necessary for the induction of complete immunotolerance. When skin transplantation was performed 7 days before liver transplantation, the grafted mouse died within 1 week after the liver transplant as a result of rejection of the liver. These results indicated that OMLT might provide
a. Specificity for class I (K) primersfor Kd t
primersfor Kk I
E. Sensitivity for class I (K)
a superior experimental model for studies designed to clarify the mechanism of graft acceptance and the induction of tolerance. Using one combination of H-2 allotypes, H-2d and H-2k, we examined microchimerism after liver transplantation in the present study. Analysis of Microchimerism
by RT-PCR
We prepared four sets of primers that we had designed for specific amplification of transcripts that encoded H-2Kk, Kd, I-Ak, and I-Ad, respectively, as indicated in Table 1. The specificity of the amplification of fragments was determined from the sizes of fragments, as indicated by their mobility on a gel. Specific amplification was observed, as shown in Fig la and lb. Digestion with restriction enzymes confirmed
b. Specificity for class II (IA) primersfor lAd I-
primersforIAk
d. Sensitivity for class II (IA)
Fig 1. Evaluation of the specificity (a, b) and sensitivity (c, d) of RT-PCR using tissues from untreated animals, namely, BlO.D2 (d-haplotype) and BlO.BR (k-haplotype). Highly specific bands at the expected position, representing transcripts of each class I and class II gene, were visualized after RT-PCR with four sets of primers. The sensitivity of RT-PCR was examined with mixtures of serially diluted samples of RNA and the limit of detection was between l/lo3 and 1/104.
MICROCHIMERISM
IN MICE
1191
P~~IIWS fix Kd prinws for KL
n-
-Kmhp ( Kd: donor ~ypc) 311 bp (K’: rccipicnt typ)
Detection of microchimerism after OMLT. A BlO.D2 (d-haplotype) graft was transplanted into a BlO.BR (k-haplotype) recipient. Fig 2. On day 14, total RNA was extracted from the spleen, thymus, and blood of the recipient. The results after RT-PCR revealed clear evidence of the transcription of both donor-type class I and class II genes, demonstrating the existence of microchimerism.
the amplification of the expected fragments (data not shown). We also examined the sensitivity of RT-PCR. Samples of RNA prepared from the liver of BlO.BR (k-haplotype) and BlO.D2 (d-haplotype) mice were mixed at ratios of 104:1 to 1:104 (w/w), and RT-PCR was performed with four sets of primers. The limit of detection was between l/10” and l/lo4 under the conditions used in this study, as shown in Fig lc and Id. The presence of transcripts that encoded donor-derived H-2K and I-A was examined in grafted mice on days 7, 14, 21,28,35, and 63. A typical example, revealing the presence of microchimerism, is shown in Fig 2. In one case, we found microchimerism of class II-expressing cells in the spleen and thymus on day 7. On day 14, all three cases examined showed microchimerism in the spleen, thymus, and blood. Moreover, in two cases, transcripts for both donor-derived class I and II molecules were detected, and in one case, only transcripts for class II molecules were detected. On day 21,
Table
2. Detection
split results were obtained. However, from day 28 to day 63, the donor-derived transcripts encoding either class I or II molecules were no longer detectable. These results are summarized in Table 2. As mentioned above, more than 30 days were required after liver transplantation for complete induction of immunotolerance to donor-type skin. Nevertheless, graft acceptance and induction of tolerance were consistently achieved regardless of the presence or absence of detectable microchimerism.
DISCUSSION
The system for OMLT has been well established in our laboratory. Successful OMLT can now be performed twice daily with a very high success rate. The results of OMLT with a large variety of allogeneic combinations of available laboratory strains indicate that spontaneous graft acceptance and induction of tolerance are general phenomena
of Microchimerism
by FIT-PCR After OMLT Microchimerism
Strain Combination Days After OMLT 7
14
21 28
Donor
Thymus
Spleen
I
Blood Class I
Class II
NT
NT NT
NT _
NT
NT NT
NT
_
_
_
_
+
NT
NT
Class I
Class II
Class I
D2
_
_
_
D2
_
+ _
NT _
NT
D2
+ _
Class
BR BR BR
Kidney Class II
Class II
Recipient
BR
D2
+
+
+
+
+
D2
BR
+
+
+
_
BR
D2
+ _
+
+ _
+
NT
NT
NT
NT
BR
D2
+
D2
+ _
+ _
NT _
NT _
+ _
+
BR
+ _
BR
D2
_
_
_
_
_
_
_
NT
NT
_
NT
NT
35
BR
D2
_
63
D2
BR
_
Abbreviation: NT, not tested.
_
_ _
_
_
1192
after OMLT. We recently used several strains of wild mice that have been established at the National Institute of Genetics at Mishima in Japan for OMLT. The results were essentially the same as those obtained with the laboratory strains and appear to be different from those obtained with rats. In the case of rat liver transplantation, three different types of results have been obtained, depending on the combination of strains.18 In one group of rats, for example, the combination of DA(RTla) as donor and PVG(RT1’) as recipient, permanent acceptance and induction of tolerance were observed consistently, as in the present study with mice. In another group of rats, for example, with DA(RTla) as donor and LEW(RTll) as recipient, the grafted rats died because of rejection about 11 days after the operation. In the third group, for example, with BN(RTlm) as donor and LEW(RT1’) as recipient, the grafted rats survived for a prolonged period but ultimately died. We confirmed these results by 600 operations for transplantation of rat liver. As long as the operation was carried out under the appropriate conditions, the above-described results were obtained consistently. Thus, the acceptance or rejection by rats of grafted livers appears to be genetically determined. The phenomenon of microchimerism after solid-organ transplantation has been well established.’ It remains unclear, however, whether the microchimerism per se is the cause of induction of tolerance or a consequence. Moreover, the detection of donor cell-derived genomic DNA by PCR that has usually been utilized to demonstrate the presence of microchimerism is too sensitive for suitable evaluation and, furthermore, such detection is not quantitative. In the present study, we used RT-PCR. Our results indicated that the number of the donor-derived MHC class II-expressing cells reached a maximum 2 weeks after liver transplantation and then decreased. Since the sensitivity of RT-PCR was between l/lo3 and 1/104, we cannot ignore the possibility that donor-derived cells remained viable for a long time. However, we can draw the following conclusions. (1) The donor-derived cells migrated throughout the recipient’s body after the liver transplantation, with microchimerism developing in many cases and possibly even in all cases. MHC class II-expressing cells that might have had antigen-presenting capability were included among the migrating cells. (2) The numbers of donor-derived cells increased 2 and 3 weeks after the operation. However, this increase in numbers of migrating cells did not seem to be essential for the induction of tolerance since the extent of the increase appeared to be variable even though the tolerance was induced in all cases. (3) Four weeks after the
SUGIOKA,
MORITA, ESAKI ET AL
operation, microchimerism, even if it remained, fell below the limit of detection by RT-PCR. (4) More than 30 days were required for the full induction of tolerance. (5) About 30 days after liver transplantation, donor-specific tolerance appeared to have been only partly induced, being related perhaps to the phenomenon known as “split tolerance.“4 (6) After prior sensitization with a skin graft, a liver graft never induced tolerance. Pluripotent hematopoietic stem cells reside in the liver.8s9 The transient appearance of donor-derived cells between 1 and 3 weeks after liver transplantation might have been due to the presence of such cells. It has been argued that the number of donor-derived cells is crucial in the deletion of donor-reactive T cells and skin graft acceptance and that there exists a threshold with respect to the extent of chimerism that is required for the induction of tolerance.19”’ Our results suggest that, although microchimerism was clearly detected, it was a transient phenomenon. Four weeks after operation when tolerance had been partially induced, the extent of microchimerism fell below the detectable level. Thus, it seems difficult to explain the induction of tolerance solely in terms of the existence of microchimerism. REFERENCES
1. Calne RY, et al: Nature 223:472, 1969
2. 3. 4. 5. 6. 7.
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