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Indirect T-cell recognition in human allograft rejection
ELSEVIER
Indirect T-Cell Recognition in Human Allograft Rejection N. Suciu-Foca, Z. Liu, A.I. Colovai, P. Fisher, E. Ho, E.F. Reed, E.A. Rose, R.E. Michler, P. Cocciolo, F. Gargano, and R. Cortesini
T
RANSPLANT rejection is mediated by T cells that recognize allogeneic MHC antigens via the direct or indirect pathway.’ The direct recognition pathway involves T cells that recognize intact allogeneic MHC/peptide complexes on the surface of donor target cells. This form of recognition does not require processing and presentation of alloantigens by host antigen presenting cells (APC) and is, therefore, not MHC-restricted. Because the frequency of T cells able to recognize alloantigens directly is very high even in nonimmunized responders, it is believed that this process reflects T-cell recognition of allogeneic MHC/peptide complexes via molecular mimicry with other antigenic structures.’ Although the majority of T cells infiltrating the graft during early acute rejection exhibit direct recognition ability, it is unlikely that these cells can mediate late or chronic rejection because their stimulation requires the presence in the graft of passenger APCs of donor origin.3 The absence of costimulatory molecules on the surface of graft endothelial and parenchimal cells renders such putative targets more likely to induce anergy rather than stimulation of recipient T lymphocytes.4 In contrast to the direct recognition pathway, indirect alloimmune responses are mediated by T cells that react
against peptides derived from the processing of allogeneic MHC proteins by host APCs.’ Peptides resulting from the proteolysis of allogeneic MHC molecules bind to MHCclass II antigens of host APCs and trigger T-cell alloimmune responses. This form of alloreactivity is restricted by host HLA-DR antigens and is carried out by an oligoclonal population of T cells, which are capable of recognizing the dominant epitope of the allogeneic MHC molecule. Because the stimulatory peptides can be generated continuously from soluble MHC alloantigens released from the graft and processed by host APC, the direct pathway may be responsible both for the initiation and perpetuation of allograft rejection. To investigate the role of the indirect recognition pathway in rejection we have monitored peripheral blood T cells from recipients of heart, renal, and liver allografts for reactivity against synthetic peptides corresponding to the hypervariable region (HVR) of donor HLA-DR antigens. We report on the significant association between T-cell alloreactivity to donor peptides and allograft rejection. 0041-l 315/97/$17.00
PII SO041 -1345(96)00347-8
MATERIALS
AND
M.A. Hardy,
METHODS
The patient population consisted of 42 heart, 35 renal, and 11 liver allograft recipients. PBMCs were obtained from each patient at the time of transplantation, and at 1 to 2 week intervals over the first 3 months following transplantation. PBMCs were cultured for 14 days in medium supplemented with IL-2 (50 U/mL), then tested in limiting dilution assays (LDA) for reactivity to synthetic allopeptides in the presence of autologous APCs. The blastogenic response was measured by 3[H]TdR incorporation after 3 days of incubation, as previously described.h All recipients and donors were typed for HLA-DR antigens by conventional serology and molecular methodology using PCRSSOP. The selection of stimulatory peptides was based on the molecular HLA-DR subtype of each individual donor. Synthetic peptides corresponding to residues l-19,21-39, and 62-80 of DRBl chain from 32 HLA-DR alleles were obtained from Chiron Mimotopes (San Diego, Calif) and Peptide Innovation (Raleigh, NC). Acute rejection of heart allografts was diagnosed on biopsies as histologic grade lB, 2, or 3A according to Billingham’s criteria.’ The diagnosis of acute rejection of renal and liver allografts was based on alterations in organ function and biopsies. Statistical analysis of the relationship between T cell reactivity to donor allopeptide and rejection was performed using BMDP Statistical Software.x RESULTS
AND
DISCUSSION
To investigate the role of indirect recognition in allograft rejection, we tested the in vitro reactivity of T cells from recipients’ peripheral blood to synthetic allopeptides corresponding to the mismatched HLA-DR antigens of the donor. LDA was considered positive when more than 1.5 X lo-” Th reactive to one or both of the donor’s HLA-DR antigens were detected. The presence of allopeptide reactive T cells was considered to correlate with rejection when LDA was positive prior to (1 to 14 days), during, or From the Departments of Pathology (NS-F., Z.L., A.I.C., P.F., E.H., E.F.R.), and Surgery (E.A.R., R.E.M., M.A.H.), College of Physicians and Surgeons of Columbia University, New York, NY; and II Clinica Chirurgica, Servizio Trapianti D’Organo, Universita’ degli Studi di Roma “La Sapienza” (PC., F.G., RD.), Roma, Italy. This work was supported in part by the National Institutes of Health Grant ROl -A12521 O-l 0. Address reprint requests to Dr Nicole Suciu-Foca, College of Physicians and Surgeons of Columbia University, Department of Pathology, 630 West 168th Street, New York, NY 10032.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1012
Transplantation
Proceedings,
29, 1012-l 013 (1997)
T-CELL RECOGNITION
1013
IN REJECTION
Table 1. Indirect Allorecognition in Heart, Kidney, and Liver Allograft Rejection
Number Number Number Positive
of Patients of LDA Determinations of Positive LDA LDA in Rejection
Heart
Kidney
32 130 51 48
17 104
ia 15
Liver
a 20 11 11
following (1 to 14 days) a rejection episode. The data in Table 1 demonstrates that in heart allograft recipients, acute rejection episodes were accompanied almost invariably by the appearance of allopeptide reactive T cells in the circulation. Activation of the indirect recognition pathway was found in 25 of 27 rejection episodes occurring in this population of heart allograft recipients. However, the response was directed against only one of the donor’s HLA-DR antigens in 21 cases. The remaining four cases, in which reactivity against both of the donor’s DR antigens was detected, represented secondary or tertiary rejection episodes. Hence, in patients with multiple rejection episodes the response expands from one of the donor HLA-DR antigens to the other, eg, there is intermolecular spreading of antigenic recognition. Studies of renal allograft recipients yielded similar results. A total of 1.5 rejection episodes were documented in a population of 17 recipients. Donor-specific T cells were present in the circulation of patients with rejection in each of these 15 cases. Nine of the patients studied were mismatched from the donor by two HLA-DR antigens. However, T-cell reactivity during rejection was directed against a single HLA-DR alloantigen of the donor in all recipients mismatched by two HLA-DR alleles. Activation of the indirect pathway was also observed in four selflimiting rejection episodes, which resolved without treatment, suggesting that allopeptide reactive T cells may be suppressed andior anergized upon exposure to soluble HLA-DR antigens released from the injured graft. Monitoring of the indirect allorecognition pathway in liver allograft recipients unraveled the presence of allopeptide reactive T cells in the circulation of seven of eight patients with acute rejection episodes. Of these eight recipients, three were mismatched from the donor by both HLA-DR antigens. Intermolecular spreading of allopeptide recognition was found in only one patient who showed T-cell reactivity against one mismatched antigen (DRll) at
the time of the first rejection episode and reactivity against the other DR allopeptides (DR7) 4 months later, when alterations in transplant function were seen. This finding is of particular importance because it indicates that the amount of soluble HLA-class II antigens released by liver allografts is not sufficient for suppressing the indirect pathway of allorecognition. Taken together, the finding that allopeptide-reactive T cells are present in the circulation of heart, kidney, and liver allograft recipients at the onset of acute rejection reveals the generality of this phenomenon and emphasizes the need for developing strategies for specific immunosuppression.
CONCLUSIONS
The present study demonstrates for the first time the role of the indirect recognition pathway in initiating acute rejection of human heart, kidney, and liver allografts. Monitoring of T-cell alloreactivity against synthetic peptides corresponding to mismatched sequences of donor HLA-DR antigens unraveled the appearance of allospecific T cells in the circulation of patients undergoing primary rejection episodes. Allopeptide-reactive T cells were not detectable during quiescence, suggesting that their activation and proliferation is causally related to the rejection process. Because T-cell reactivity to donor alloantigens is directed against a single dominant alloepitope, the indirect pathway of allorecognition may be amenable to specific suppression by induction of high zone tolerance or by use of altered peptide ligands with antagonist function.
REFERENCES
1. Sherman LA, Chattopadhyan S: Ann Rev Immunol 11:385, 1993 2. Lechler RI, Heaton T, Barber L, et al: Immunol Lett 34:63, 1992 3. Lechler RI, Batchelor JR: J Exp Med 155:31, 1982 4. Gimmi CD, Freeman GJ, Gribben JF, et al: Proc Nat1 Acad Sci USA 90:6586, 1993 5. Bradley JA: Int Rev Immunol 13:245, 1996 6. Liu Z, Colovai AI, Tugulea S, et al: J Clin Invest 98:1150,1996 7. Billingham MD: Williams and Wilkins, Baltimore MD. 108: 1995 8. Dixon WJ, Brown MB, Engelman L, et al: BMDP statistical software manual to accompany the 1990 software release, Berkeley University of California Press
Indirect T-Cell Recognition in Human Allograft Rejection N. Suciu-Foca, Z. Liu, A.I. Colovai, P. Fisher, E. Ho, E.F. Reed, E.A. Rose, R.E. Michler, P. Cocciolo, F. Gargano, and R. Cortesini
T
RANSPLANT rejection is mediated by T cells that recognize allogeneic MHC antigens via the direct or indirect pathway.’ The direct recognition pathway involves T cells that recognize intact allogeneic MHC/peptide complexes on the surface of donor target cells. This form of recognition does not require processing and presentation of alloantigens by host antigen presenting cells (APC) and is, therefore, not MHC-restricted. Because the frequency of T cells able to recognize alloantigens directly is very high even in nonimmunized responders, it is believed that this process reflects T-cell recognition of allogeneic MHC/peptide complexes via molecular mimicry with other antigenic structures.’ Although the majority of T cells infiltrating the graft during early acute rejection exhibit direct recognition ability, it is unlikely that these cells can mediate late or chronic rejection because their stimulation requires the presence in the graft of passenger APCs of donor origin.3 The absence of costimulatory molecules on the surface of graft endothelial and parenchimal cells renders such putative targets more likely to induce anergy rather than stimulation of recipient T lymphocytes.4 In contrast to the direct recognition pathway, indirect alloimmune responses are mediated by T cells that react
against peptides derived from the processing of allogeneic MHC proteins by host APCs.’ Peptides resulting from the proteolysis of allogeneic MHC molecules bind to MHCclass II antigens of host APCs and trigger T-cell alloimmune responses. This form of alloreactivity is restricted by host HLA-DR antigens and is carried out by an oligoclonal population of T cells, which are capable of recognizing the dominant epitope of the allogeneic MHC molecule. Because the stimulatory peptides can be generated continuously from soluble MHC alloantigens released from the graft and processed by host APC, the direct pathway may be responsible both for the initiation and perpetuation of allograft rejection. To investigate the role of the indirect recognition pathway in rejection we have monitored peripheral blood T cells from recipients of heart, renal, and liver allografts for reactivity against synthetic peptides corresponding to the hypervariable region (HVR) of donor HLA-DR antigens. We report on the significant association between T-cell alloreactivity to donor peptides and allograft rejection. 0041-l 315/97/$17.00
PII SO041 -1345(96)00347-8
MATERIALS
AND
M.A. Hardy,
METHODS
The patient population consisted of 42 heart, 35 renal, and 11 liver allograft recipients. PBMCs were obtained from each patient at the time of transplantation, and at 1 to 2 week intervals over the first 3 months following transplantation. PBMCs were cultured for 14 days in medium supplemented with IL-2 (50 U/mL), then tested in limiting dilution assays (LDA) for reactivity to synthetic allopeptides in the presence of autologous APCs. The blastogenic response was measured by 3[H]TdR incorporation after 3 days of incubation, as previously described.h All recipients and donors were typed for HLA-DR antigens by conventional serology and molecular methodology using PCRSSOP. The selection of stimulatory peptides was based on the molecular HLA-DR subtype of each individual donor. Synthetic peptides corresponding to residues l-19,21-39, and 62-80 of DRBl chain from 32 HLA-DR alleles were obtained from Chiron Mimotopes (San Diego, Calif) and Peptide Innovation (Raleigh, NC). Acute rejection of heart allografts was diagnosed on biopsies as histologic grade lB, 2, or 3A according to Billingham’s criteria.’ The diagnosis of acute rejection of renal and liver allografts was based on alterations in organ function and biopsies. Statistical analysis of the relationship between T cell reactivity to donor allopeptide and rejection was performed using BMDP Statistical Software.x RESULTS
AND
DISCUSSION
To investigate the role of indirect recognition in allograft rejection, we tested the in vitro reactivity of T cells from recipients’ peripheral blood to synthetic allopeptides corresponding to the mismatched HLA-DR antigens of the donor. LDA was considered positive when more than 1.5 X lo-” Th reactive to one or both of the donor’s HLA-DR antigens were detected. The presence of allopeptide reactive T cells was considered to correlate with rejection when LDA was positive prior to (1 to 14 days), during, or From the Departments of Pathology (NS-F., Z.L., A.I.C., P.F., E.H., E.F.R.), and Surgery (E.A.R., R.E.M., M.A.H.), College of Physicians and Surgeons of Columbia University, New York, NY; and II Clinica Chirurgica, Servizio Trapianti D’Organo, Universita’ degli Studi di Roma “La Sapienza” (PC., F.G., RD.), Roma, Italy. This work was supported in part by the National Institutes of Health Grant ROl -A12521 O-l 0. Address reprint requests to Dr Nicole Suciu-Foca, College of Physicians and Surgeons of Columbia University, Department of Pathology, 630 West 168th Street, New York, NY 10032.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1012
Transplantation
Proceedings,
29, 1012-l 013 (1997)
T-CELL RECOGNITION
1013
IN REJECTION
Table 1. Indirect Allorecognition in Heart, Kidney, and Liver Allograft Rejection
Number Number Number Positive
of Patients of LDA Determinations of Positive LDA LDA in Rejection
Heart
Kidney
32 130 51 48
17 104
ia 15
Liver
a 20 11 11
following (1 to 14 days) a rejection episode. The data in Table 1 demonstrates that in heart allograft recipients, acute rejection episodes were accompanied almost invariably by the appearance of allopeptide reactive T cells in the circulation. Activation of the indirect recognition pathway was found in 25 of 27 rejection episodes occurring in this population of heart allograft recipients. However, the response was directed against only one of the donor’s HLA-DR antigens in 21 cases. The remaining four cases, in which reactivity against both of the donor’s DR antigens was detected, represented secondary or tertiary rejection episodes. Hence, in patients with multiple rejection episodes the response expands from one of the donor HLA-DR antigens to the other, eg, there is intermolecular spreading of antigenic recognition. Studies of renal allograft recipients yielded similar results. A total of 1.5 rejection episodes were documented in a population of 17 recipients. Donor-specific T cells were present in the circulation of patients with rejection in each of these 15 cases. Nine of the patients studied were mismatched from the donor by two HLA-DR antigens. However, T-cell reactivity during rejection was directed against a single HLA-DR alloantigen of the donor in all recipients mismatched by two HLA-DR alleles. Activation of the indirect pathway was also observed in four selflimiting rejection episodes, which resolved without treatment, suggesting that allopeptide reactive T cells may be suppressed andior anergized upon exposure to soluble HLA-DR antigens released from the injured graft. Monitoring of the indirect allorecognition pathway in liver allograft recipients unraveled the presence of allopeptide reactive T cells in the circulation of seven of eight patients with acute rejection episodes. Of these eight recipients, three were mismatched from the donor by both HLA-DR antigens. Intermolecular spreading of allopeptide recognition was found in only one patient who showed T-cell reactivity against one mismatched antigen (DRll) at
the time of the first rejection episode and reactivity against the other DR allopeptides (DR7) 4 months later, when alterations in transplant function were seen. This finding is of particular importance because it indicates that the amount of soluble HLA-class II antigens released by liver allografts is not sufficient for suppressing the indirect pathway of allorecognition. Taken together, the finding that allopeptide-reactive T cells are present in the circulation of heart, kidney, and liver allograft recipients at the onset of acute rejection reveals the generality of this phenomenon and emphasizes the need for developing strategies for specific immunosuppression.
CONCLUSIONS
The present study demonstrates for the first time the role of the indirect recognition pathway in initiating acute rejection of human heart, kidney, and liver allografts. Monitoring of T-cell alloreactivity against synthetic peptides corresponding to mismatched sequences of donor HLA-DR antigens unraveled the appearance of allospecific T cells in the circulation of patients undergoing primary rejection episodes. Allopeptide-reactive T cells were not detectable during quiescence, suggesting that their activation and proliferation is causally related to the rejection process. Because T-cell reactivity to donor alloantigens is directed against a single dominant alloepitope, the indirect pathway of allorecognition may be amenable to specific suppression by induction of high zone tolerance or by use of altered peptide ligands with antagonist function.
REFERENCES
1. Sherman LA, Chattopadhyan S: Ann Rev Immunol 11:385, 1993 2. Lechler RI, Heaton T, Barber L, et al: Immunol Lett 34:63, 1992 3. Lechler RI, Batchelor JR: J Exp Med 155:31, 1982 4. Gimmi CD, Freeman GJ, Gribben JF, et al: Proc Nat1 Acad Sci USA 90:6586, 1993 5. Bradley JA: Int Rev Immunol 13:245, 1996 6. Liu Z, Colovai AI, Tugulea S, et al: J Clin Invest 98:1150,1996 7. Billingham MD: Williams and Wilkins, Baltimore MD. 108: 1995 8. Dixon WJ, Brown MB, Engelman L, et al: BMDP statistical software manual to accompany the 1990 software release, Berkeley University of California Press