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Mechanism of liver allograft rejection: indirect allorecognition
Mechanism of Liver Allograft Rejection: Indirect Allorecognition E. Renna-Molajoni, P. Cinti, L. Elia, A.M. Orlandini, P. Cocciolo, J. Molajoni, E. Ho, N. Suciu-Foca, and R. Cortesini
D
ESPITE NUMEROUS studies, the mechanism that underlies the rejection or acceptance of liver allografts is not completely understood. There is evidence that HLA matching has a relatively weak “dualistic effect” reflecting, on one hand, the increased incidence of rejection in the presence of major histocompatibility complex (MHC) incompatibility and, on the other hand, the augmented rate of immunologic injury (caused by recurrence of the original disease) in patients with HLA-matched liver allografts.1– 6 Furthermore, MHC Class I antigens secreted or shed by liver allografts may have a tolerogenic effect.7 In contrast, the expression of MHC Class II antigens on vascular endothelial and biliary epithelium cells is upregulated as a consequence of immunologic injury, and may contribute to the induction of T cell reactivity rather than unresponsiveness to the graft.8 However, because neither endothelial nor biliary epithelial cells express the B7 molecules required for efficient costimulation of alloreactive T cells, the direct pathway of allorecognition cannot explain the development of rejection after the departure of donor dendritic cells from the graft. Furthermore, the diagnosis of acute rejection is more difficult to ascertain at later periods because the differential diagnosis includes post-transplantation lymphoproliferative disorders, recurrent viral hepatitis type B or C, recurrent primary biliary cirrhosis, or autoimmune hepatitis and obstructive cholangiopathy.1 Similar difficulties are encountered in the early diagnosis of chronic rejection which can develop after unresolved episode(s) of acute rejection or indolently in patients with no clinically apparent episodes of acute rejection. In this study we have explored the contribution of the indirect allorecognition pathway to liver allograft rejection. Our data show that T cell reactivity to donor allopeptides is predictive of acute rejection permitting early identification of patients at risk. Furthermore, based on the recent characterization of molecular events associated with suppression, we have developed a strategy for identifying patients who have developed such regulatory T cells and are likely to remain in quiescence. MATERIALS AND METHODS Peripheral blood mononuclear cells (PBMC) were obtained from 23 patients who underwent orthotopic liver transplantation at the University of Rome “La Sapienza.” PBMCs were tested for © 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
reactivity to synthetic peptides corresponding to the hypervariable region of donor HLA-DR antigens. The precursor frequency of allopeptide reactive T cells in the patients’ circulation was determined as previously described using sequential samples of blood obtained after 2 to 575 days following transplantation.9 The methods used for diagnosis and treatment of acute rejection as well as for statistical analysis of the association between rejection and allopeptide reactivity were described before.9 The method used for detection of suppressor cells as well as follows. Patients’ PBMCs were separated into CD41 and CD81 subsets using CD4 and CD8 Dynal beads. Donor B cells (1 3 106/mL) were obtained from cryopreserved spleen cell suspensions using Dynalbeads coupled with CD20 and detachabeads. Cultures containing donor B cells alone, B cells and CD41 T cells, B cells and CD81 T cells, and B cells plus both CD41 and CD81 T cells were set up in parallel. The T to B cell ratio was kept constant at 1:1. After 24 hours of incubation at 37°C cultures were stained with CD20 Per Cp, CD80 FITC, and CD86 PE. The expression of CD80 and CD86 on CD201 B cells was determined on a FacScan Flow Cytometer (Becton Dickinson) as described.10
RESULTS AND DISCUSSIONS
To determine the predictive value of allopeptide reactivity in acute rejection we monitored a population of 23 patients for periods of 6 to 82 weeks after transplantation. Table 1 shows the relationship between the results of 97 LDA determinations of allopeptide reactivity in this population and the development of acute rejection episodes within 1 month after the performance of the test. The cumulative incidence of acute rejection episodes occurring within 7 to 30 days after a positive LDA result was 91%. The corresponding frequency in patients without detectable antidonor reactivity was 27%. The difference between the groups was highly significant (P , .0001), From ISMEDA-CNR (E.R.-M., A.M.O., J.M.), Italy; II Clinica Chirurgica (P.Ci, P.Co., R.C.), Dep. Biotecnologie Cellulari ed Ematologia (L.E.), Universita di Roma “La Sapienza,” Servizio Trapianti d’Organo, Rome, Italy; and the College of Physicians and Surgeons of Columbia University (E.H., N.S.-F.), Department of Pathology, New York, N.Y. This work was supported by a grant from the National Institute of Health 5-RO1A125210-11 and by the Consorzio Interuniversitario Trapianti d’Organo, Rome, Italy. Address reprint requests to Dr E Molajoni, II Clinica Chirurgica, Policlinico Umberto I, Viale Del Policlinico, 00161 Roma, Italia. 0041-1345/99/$–see front matter PII S0041-1345(98)01683-2 409
Transplantation Proceedings, 31, 409–410 (1999)
410
RENNA-MOLAJONI, CINTI, ELIA ET AL
Table 1. Relationship Between Acute Rejection and Th Reactivity to Donor Peptides in Liver Allograft Recipients
Table 3. Relationship Between the Presence of T Suppressor Cells and Acute Rejection Acute rejection
Cumulative Incidence of Acute Rejection Days After LDA
LDA1 (n 5 52)
LDA2 (n 5 43)
0 7 14 21 30
0% 81% 85% 85% 91%
0% 10% 15% 15% 27%
Ts1 Ts22
indicating that allopeptide reactivity is predictive of liver allograft rejection. To determine whether persistent allopeptide reactivity reflects an ongoing immune response, eventually leading to multiple acute rejection episodes or chronic rejection, we analyzed the clinical outcome of patients who showed donor-reactive T cells in more than 25% of serial determinations (N 5 19). There was a highly significant correlation between persistent alloreactivity and multiple or chronic rejection (P , .007). Twelve of 23 patients included in this study were tested for the presence of T cells in the peripheral blood 6 months to 2 years after transplantation. The test was based on a previous study which showed that allospecific CD81 T cells inhibit Th reactivity against the specific target by downregulating the expression of the CD80 and CD86 molecules, required for Th costimulation.10 Six of seven patients tested had one or no rejection episodes. In contrast, all five patients without T cells had Table 2. Relationship Between Chronic and Multiple Acute Rejections (CR/MR) and Persistent Allopeptide Reactivity in LDA CR/MR 1
2
Total
15 0 15
4 4 8
19 4 23
Fisher Exact Test (2-tails): P , .007.
.1
Total
6 1 7
0 5 5
6 6 12
Fisher Exact Test (2-tails): P , .01.
Cumulative incidence was calculated by the product-limit method. The difference between presence and absence of Th reactivity was compared using Breslow statistics (P , .0001). Abbreviation: LDA.
LDA1 LDA2
#1
multiple acute rejection episodes. This indicates that there is an inverse correlation between T cells and acute rejection (P , .02). Taken together, these data show that liver allograft rejection is mediated by Th cells which react to HLA-DR allopeptides processed and presented by host antigen presenting cells (APCs). Allopeptide-reactive Th provide the cytokines required for upregulation of B7 (CD80 and 86) expression on host APCs. This may result in the transstimulation of T cells with direct recognition ability enabling them to attack the vascular endothelium and the biliary epithelium. Maintenance of allograft tolerance, therefore, is an active process requiring inhibition of allopeptide reactivity and activation of antigen-specific regulatory T cells. REFERENCES 1. Demetris A: Mayo Clin Proc 73:367, 1998 2. Markus BH, Fung JJ, Gordon RD, et al: Transplant Proc 19:63, 1987 3. Markus BH, Duquesnoy RJ, Gordon RD, et al: Transplantation 46:372, 1988 4. Starzi TE, Demetris AJ, Trucco M: Transplant Proc 25:2450, 1993 5. Donaldson P, Underhill J, Doherty D, et al: Hepatology 17:1008, 1993 6. Terasaki PI (ed.) Clinical Transplants 1994. Los Angeles, CA; UCLA Tissue Typing Laboratory; 1994 7. Davies HS, Pollard SG, Calne RY: Transplantation 47:524, 1989 8. Leon MP, Bassendine MF, Gibbs P, et al: Gastroenterology 112:968, 1997 9. Renna-Molajoni E, Cinti P, Orlandini A, et al: Human Immunol 53:57, 1997 10. Liu Z, Tugulea S, Cortesini R, et al: Int Immunol 10:101, 1998
D
ESPITE NUMEROUS studies, the mechanism that underlies the rejection or acceptance of liver allografts is not completely understood. There is evidence that HLA matching has a relatively weak “dualistic effect” reflecting, on one hand, the increased incidence of rejection in the presence of major histocompatibility complex (MHC) incompatibility and, on the other hand, the augmented rate of immunologic injury (caused by recurrence of the original disease) in patients with HLA-matched liver allografts.1– 6 Furthermore, MHC Class I antigens secreted or shed by liver allografts may have a tolerogenic effect.7 In contrast, the expression of MHC Class II antigens on vascular endothelial and biliary epithelium cells is upregulated as a consequence of immunologic injury, and may contribute to the induction of T cell reactivity rather than unresponsiveness to the graft.8 However, because neither endothelial nor biliary epithelial cells express the B7 molecules required for efficient costimulation of alloreactive T cells, the direct pathway of allorecognition cannot explain the development of rejection after the departure of donor dendritic cells from the graft. Furthermore, the diagnosis of acute rejection is more difficult to ascertain at later periods because the differential diagnosis includes post-transplantation lymphoproliferative disorders, recurrent viral hepatitis type B or C, recurrent primary biliary cirrhosis, or autoimmune hepatitis and obstructive cholangiopathy.1 Similar difficulties are encountered in the early diagnosis of chronic rejection which can develop after unresolved episode(s) of acute rejection or indolently in patients with no clinically apparent episodes of acute rejection. In this study we have explored the contribution of the indirect allorecognition pathway to liver allograft rejection. Our data show that T cell reactivity to donor allopeptides is predictive of acute rejection permitting early identification of patients at risk. Furthermore, based on the recent characterization of molecular events associated with suppression, we have developed a strategy for identifying patients who have developed such regulatory T cells and are likely to remain in quiescence. MATERIALS AND METHODS Peripheral blood mononuclear cells (PBMC) were obtained from 23 patients who underwent orthotopic liver transplantation at the University of Rome “La Sapienza.” PBMCs were tested for © 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
reactivity to synthetic peptides corresponding to the hypervariable region of donor HLA-DR antigens. The precursor frequency of allopeptide reactive T cells in the patients’ circulation was determined as previously described using sequential samples of blood obtained after 2 to 575 days following transplantation.9 The methods used for diagnosis and treatment of acute rejection as well as for statistical analysis of the association between rejection and allopeptide reactivity were described before.9 The method used for detection of suppressor cells as well as follows. Patients’ PBMCs were separated into CD41 and CD81 subsets using CD4 and CD8 Dynal beads. Donor B cells (1 3 106/mL) were obtained from cryopreserved spleen cell suspensions using Dynalbeads coupled with CD20 and detachabeads. Cultures containing donor B cells alone, B cells and CD41 T cells, B cells and CD81 T cells, and B cells plus both CD41 and CD81 T cells were set up in parallel. The T to B cell ratio was kept constant at 1:1. After 24 hours of incubation at 37°C cultures were stained with CD20 Per Cp, CD80 FITC, and CD86 PE. The expression of CD80 and CD86 on CD201 B cells was determined on a FacScan Flow Cytometer (Becton Dickinson) as described.10
RESULTS AND DISCUSSIONS
To determine the predictive value of allopeptide reactivity in acute rejection we monitored a population of 23 patients for periods of 6 to 82 weeks after transplantation. Table 1 shows the relationship between the results of 97 LDA determinations of allopeptide reactivity in this population and the development of acute rejection episodes within 1 month after the performance of the test. The cumulative incidence of acute rejection episodes occurring within 7 to 30 days after a positive LDA result was 91%. The corresponding frequency in patients without detectable antidonor reactivity was 27%. The difference between the groups was highly significant (P , .0001), From ISMEDA-CNR (E.R.-M., A.M.O., J.M.), Italy; II Clinica Chirurgica (P.Ci, P.Co., R.C.), Dep. Biotecnologie Cellulari ed Ematologia (L.E.), Universita di Roma “La Sapienza,” Servizio Trapianti d’Organo, Rome, Italy; and the College of Physicians and Surgeons of Columbia University (E.H., N.S.-F.), Department of Pathology, New York, N.Y. This work was supported by a grant from the National Institute of Health 5-RO1A125210-11 and by the Consorzio Interuniversitario Trapianti d’Organo, Rome, Italy. Address reprint requests to Dr E Molajoni, II Clinica Chirurgica, Policlinico Umberto I, Viale Del Policlinico, 00161 Roma, Italia. 0041-1345/99/$–see front matter PII S0041-1345(98)01683-2 409
Transplantation Proceedings, 31, 409–410 (1999)
410
RENNA-MOLAJONI, CINTI, ELIA ET AL
Table 1. Relationship Between Acute Rejection and Th Reactivity to Donor Peptides in Liver Allograft Recipients
Table 3. Relationship Between the Presence of T Suppressor Cells and Acute Rejection Acute rejection
Cumulative Incidence of Acute Rejection Days After LDA
LDA1 (n 5 52)
LDA2 (n 5 43)
0 7 14 21 30
0% 81% 85% 85% 91%
0% 10% 15% 15% 27%
Ts1 Ts22
indicating that allopeptide reactivity is predictive of liver allograft rejection. To determine whether persistent allopeptide reactivity reflects an ongoing immune response, eventually leading to multiple acute rejection episodes or chronic rejection, we analyzed the clinical outcome of patients who showed donor-reactive T cells in more than 25% of serial determinations (N 5 19). There was a highly significant correlation between persistent alloreactivity and multiple or chronic rejection (P , .007). Twelve of 23 patients included in this study were tested for the presence of T cells in the peripheral blood 6 months to 2 years after transplantation. The test was based on a previous study which showed that allospecific CD81 T cells inhibit Th reactivity against the specific target by downregulating the expression of the CD80 and CD86 molecules, required for Th costimulation.10 Six of seven patients tested had one or no rejection episodes. In contrast, all five patients without T cells had Table 2. Relationship Between Chronic and Multiple Acute Rejections (CR/MR) and Persistent Allopeptide Reactivity in LDA CR/MR 1
2
Total
15 0 15
4 4 8
19 4 23
Fisher Exact Test (2-tails): P , .007.
.1
Total
6 1 7
0 5 5
6 6 12
Fisher Exact Test (2-tails): P , .01.
Cumulative incidence was calculated by the product-limit method. The difference between presence and absence of Th reactivity was compared using Breslow statistics (P , .0001). Abbreviation: LDA.
LDA1 LDA2
#1
multiple acute rejection episodes. This indicates that there is an inverse correlation between T cells and acute rejection (P , .02). Taken together, these data show that liver allograft rejection is mediated by Th cells which react to HLA-DR allopeptides processed and presented by host antigen presenting cells (APCs). Allopeptide-reactive Th provide the cytokines required for upregulation of B7 (CD80 and 86) expression on host APCs. This may result in the transstimulation of T cells with direct recognition ability enabling them to attack the vascular endothelium and the biliary epithelium. Maintenance of allograft tolerance, therefore, is an active process requiring inhibition of allopeptide reactivity and activation of antigen-specific regulatory T cells. REFERENCES 1. Demetris A: Mayo Clin Proc 73:367, 1998 2. Markus BH, Fung JJ, Gordon RD, et al: Transplant Proc 19:63, 1987 3. Markus BH, Duquesnoy RJ, Gordon RD, et al: Transplantation 46:372, 1988 4. Starzi TE, Demetris AJ, Trucco M: Transplant Proc 25:2450, 1993 5. Donaldson P, Underhill J, Doherty D, et al: Hepatology 17:1008, 1993 6. Terasaki PI (ed.) Clinical Transplants 1994. Los Angeles, CA; UCLA Tissue Typing Laboratory; 1994 7. Davies HS, Pollard SG, Calne RY: Transplantation 47:524, 1989 8. Leon MP, Bassendine MF, Gibbs P, et al: Gastroenterology 112:968, 1997 9. Renna-Molajoni E, Cinti P, Orlandini A, et al: Human Immunol 53:57, 1997 10. Liu Z, Tugulea S, Cortesini R, et al: Int Immunol 10:101, 1998