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Development of anti-HLA-antibodies against intra-CREG-mismatches in renal transplant recipients
Development of Anti-HLA-Antibodies Against Intra-CREG-Mismatches in Renal Transplant Recipients A.C. Papassavas, A. Iniotaki-Theodoraki, J. Boletis, A. Kostakis, and C. Stavropoulos-Giokas
C
LASS-I AND MANY class-II HLA gene products bear multiple immunogenic alloepitopes (private and public epitopes) that are differentially shared among various class I and class II HLA-molecules. Each private determinant represents a unique epitope configuration on one HLA molecule, whereas a public determinant represents an epitope shared by more than one HLA molecule. The shared or public epitopes have been used to define the major serological crossreactive epitope clusters groups (CREGs).1,2 Antibodies directed against epitopes of the HLA-antigens can be categorized according to specificity toward private and public determinants. Nearly 90% of sera from sensitized patients contain antibodies with specificity to public or crossreactive groups (CREGs) epitopes.3 Furthermore, it has been published that sensitized and highly sensitized patients do not usually develop anti-HLA-antibodies specific to antigens that belong to their own CREGphenotype.3,4 This observation could support the hypothesis that the acceptable mismatched antigens in sensitized patients belong to the same CREG with the recipients CREG-phenotype. To analyze the previous possibility, we evaluated the specificity of anti-HLA-antibodies directed against intraCREG-mismatches (intra-CREG-MMs) in alloimmunized patients.
rized as intra-CREGs-MMs and other-CREGs-MMs. The same classification was used for the specificities of the detected anti-HLA-antibodies. Analysis of anti-HLA specificity. Antibody specificity of the AHG-CDC data was based on 2 3 2 table analysis using chi square statistics to determine significant correlations between serum reactivity patterns and the presence of specific markers in the cell panel. In addition, the SOFTSTAT program analysis data was used for the definition of the antibody specificity detected by ELISA. The SOFTSTAT program performs analysis of antibody specificity, by the Fisher Exact Test. RESULTS
Forty-nine patients (49/50, 98%) had received grafts with at least one class I HLA-mismatched-antigen (Table 1) and in 63.2% (31/49) were found intra-CREGs-MMs. Thirty-six patients developed anti-HLA-antibodies. Antibodies with intra-CREGs-specificity were detected in 29% (9/31) of the patients (Table 1). All these anti-HLA-antibodies were donor specific. Antibodies with other-CREGs-MMs were detected in 37/42 (88%) cases. Twenty-six (26/37, 70%) of these anti-HLA-antibodies were donor specific (Table 1). In Table 2 is shown the HLA-specificity of the anti-HLAantibodies in the screening panel, which is compared with the intra-CREG-MMs in nine patients.
MATERIALS AND METHODS The study group consisted of 50 patients (33 males and 17 females), mean age 46 6 8 years. Thirty one (31) patients had a previous renal graft loss, and nineteen (19) patients were renal transplant recipients with biopsy proven chronic rejection (CR). Three hundred ten (310) serum samples collected prior transplantation, at various times posttransplantation, and after the graft loss in the 31 patients, were tested for anti-HLA-antibodies. The %PRA and the specificity of anti-HLA-antibodies were studied by complement dependent cytotoxicity (CDC) and Elisa technique. Cytotoxicity testing for class I anti-HLA-Abs was performed on T lymphocytes from 60 HLA-phenotyped donors using the anti-human globulin (AHG-CDC) augmented modification of the CDC technique.5,6 The PRA-STAT test (SangStat Medical Corp.) was performed according to the manufacturer’s instructions.7
HLA-phenotype. All recipients and donors were typed for class I HLA-antigens by a standard lymphocytotoxicity technique. The mismatched HLA-antigens were catego© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
DISCUSSION
It is generally accepted that humoral immunity plays a prominent role in graft rejection. This became apparent when preformed recipient antidonor anti-HLA-antibodies were shown to cause hyperacute rejection.8 In contrast, the data concerning the relevance of anti-HLA-antibodies appearing posttransplant are more controversial. Some studies have shown that the production of posttransplant antiFrom the Department of Immunology and National Tissue Typing Center, General Hospital of Athens “Georgios Gennimatas,” Department of Nephrology, and Transplant Unit, Laikon General Hospital, Athens, Greece. Address reprint requests to Andreas Papassavas, PhD, Department of Immunology and National Tissue Typing Center, General Hospital of Athens “Georgios Gennimatas,” Mesogion 154 Ave, 11527, Athens, Greece. E-mail: [email protected]. 0041-1345/99/$–see front matter PII S0041-1345(98)01751-5 757
Transplantation Proceedings, 31, 757–759 (1999)
758
PAPASSAVAS, INIOTAKI–THEODORAKI, BOLETIS ET AL
Table 1. Evaluation of the Donor CREGs-Mismatches and the Donor-Specific Class I Anti-HLA-Antibodies in 49 Renal Transplant Recipients
Graft mismatches 1. Only intra-CREGs-mismatches Anti-HLA-antibodies formation Donor specific Without anti-HLA-antibodies 2. Other and intra-CREGs-mismatches* Anti-HLA-antibodies formation Donor specific intra-CREGs-MMs Donor specific other-CREGs-MMs Non donor specific other-CREGsMMs With specificity against other-non MMs-CREGs Without anti-HLA-antibodies
n
%
49 7
98 14.29
4 3 42
57.14 42.86 85.71
5 26 3
11.9 61.9 7.14
8 10
19 23.8
*Twenty-four patients had both intra- and other-CREG-MMs and 18 patients had only other-CREG-MMs.
HLA-antibodies is associated with poor graft outcome,9,10 and anti-HLA-antibodies have been eluted from rejected kidney allografts.11,12 Moreover, it has been established that preformed IgG anti-HLA-antibodies specific for donor HLA-antigens may result in accelerated graft failure.8 However, other data are contradictory, and the relevance of posttransplant anti-HLA-antibodies remains to be defined.13 It is obvious that the detection of PRA after
transplantation can act only as an indicator of anti-donor reactivity in the recipient and that the detected specificity of the anti-HLA-antibodies, rather than isotype, is of key importance.14,15 The results of this study support the observation that graft recipients perform antibodies against their own CREG mismatched antigens in a smaller degree, compared to other CREG mismatched antigens (29% vs 70%). The observation that all these antibodies were donor specific, and possibly directed against private epitopes of the donor HLA-mismatched antigens, is of much interest. This observation could be explained by the hypothesis that HLAantigens that belong to the same CREG carry both public and private epitopes. Alloreactivity or non–reactivity of recipients immune system depends on which epitope is presented to the alloreactive T cells. The immunogenic private epitopes are presented to specific alloreactive T cells with consequence lymphokine secretion providing help for allontibody production. Public and possibly nonimmunogenic epitopes presented to recipients T cells produce tolerance rather than reactivity. Consequently, a matching strategy based on the detection of acceptable mismatches, which belong to the recipient own CREG-phenotype could be used in order to increase the pool of the most suitable donors,16 although this strategy seems not to be adequate enough to replace the conventional HLA-matching. Thus, the epitope analysis of the anti-HLA-antibodies with intra-
Table 2. HLA-Specificity Analysis of the Anti-HLA-Antibodies in the Screening Panel and Comparison With the Intra-CREGs-MMs Patients (n 5 50)
Patient HLA-phenotype
1.
A2, A11, B7, B18, DR8, DR11
2C, 1C, 7C, 8C, 6C
2.
A23, A28, B44, B51, DR2, DR5
1C, 2C, 12C, 5C, 4C
3.
A24, A28, B18, Bx, DR11, DR16
1C, 2C, 8C, 6C
4.
A24, Ax, B13, B35, DR4, DR7
1C, 7C, 5C, 4C, 6C
5.
A2, A11, B44, B60, DR1, DR5
2C, 1C, 12C, 7C, 4C, 6C
6.
A24, A28, B18, B39, DR2, DR5
1C, 2C, 8C, 6C
7.
A2, A30, B13, B51, DR10, DR11 A1, A26, B40, B51, DR5, DR14
2C, 1C, 7C, 5C, 4C 1C, 10C, 7C, 5C, 6C, 4C
A26, A28, B35, B51, DR1, DR13
10C, 2C, 5C, 4C, 6C
8.
9.
Patient CREGs
Donor HLA-phenotype
A24, A29, B7, B8, DR10, DR11 A1, Ax, B52, B57, DR2, DR9 A2, A23, B18, B14, DR11, DR13 A2, A3, B13, B35, DR2, DR7 A2, A3, B7, B44, DR5, DRx A1, A2, B18, B57, DR2, DR5 A1, A2, B8, B51, DR2, DR10 A1, A23, B51, Bx, DR5, DRx A2, B18, B57, DR5, DR7
Intra CREG-MMs
Intra CREGs
1C, 7C, 8C, 6C
A24
1C
A24
1C
1C, 5C, 21C, 4C
A1
1C
A1
1C, 21C
2C, 1C, 8C, 6C
A2, A23
2C, 1C
A2
2C
2C, 1C, 7C, 5C, 4C, 6C
A3
1C
A3
1C
2C, 1C, 7C, 12C, 6C, 4C 1C, 2C, 8C, 21C, 6C, 4C 1C, 2C, 8C, 5C, 6C, 4C
A3, B7
1C, 7C
B7
7C
A1, A2
1C, 2C
A1
1C
A1
1C
A1
1C
1C, 5C, 4C
A23
1C
A23
1C
2C, 8C, 21C, 4C, 6C
A2
2C
A2
2C
Donor CREGs
Anti-HLA Specificity
Anti-HLA CREGs
DEVELOPMENT OF ANTI-HLA-ANTIBODIES
CREG-specificity could be useful for the detection of the non-acceptable intra-CREG-antigens. REFERENCES 1. Rodey GE, Fuller TC: CRC Ctic Rev Immunol 7:229, 1987 2. Fuller TC: Clin Lab Med 11:551, 1991 3. Rodey GE, Neylan JF, Whelchel JD, et al: Hum Immunol 39:272, 1994 4. Delmonico FL, Fuller A, Cosimi AB, et al: Transplantation 36:629, 1983 5. Johnson AH, Rossen RD, and Butler WT: Tissue Antigens 2:215, 1972 6. Hopkins KA: In Zachary AA, Terisi GA (eds): ASHI Laboratory Manual, 2nd ed. 1990, p. 195 7. Zachary AA, Griffin J, Lucas DP, et al: Transplantation 60:1600, 1995
759 8. Kissmeyer-Nielsen F, Olsen S, Petersen VP, et al: Lancet 1:662, 1966 9. Nunez G, McPhaul JR, Stastny P: Transplantation 35:446, 1983 10. London RE, Stelzer GT, Ferrone S: Transplantation 38:493, 1984 11. Vogeler U, Doxiadis I, Eigler FW, et al: Transplant Proc 17:2630, 1985 12. Martin S, Dyer P, Mallick NP, et al: Transplantation 44:50, 1987 13. Ting A, Morris PI: Transplantation 28:13, 1979 14. Worthington JE, Thomas AA, Dyer PA, et al: Transplantation 65:121, 1998 15. Rodey GE, Revels K, and Fuller TC: Transplantation 63: 885, 1997 16. Papassavas AC, Iniotaki-Theodoraki A, Boletis J, et al: Hum Immunol 59:587, 1998
C
LASS-I AND MANY class-II HLA gene products bear multiple immunogenic alloepitopes (private and public epitopes) that are differentially shared among various class I and class II HLA-molecules. Each private determinant represents a unique epitope configuration on one HLA molecule, whereas a public determinant represents an epitope shared by more than one HLA molecule. The shared or public epitopes have been used to define the major serological crossreactive epitope clusters groups (CREGs).1,2 Antibodies directed against epitopes of the HLA-antigens can be categorized according to specificity toward private and public determinants. Nearly 90% of sera from sensitized patients contain antibodies with specificity to public or crossreactive groups (CREGs) epitopes.3 Furthermore, it has been published that sensitized and highly sensitized patients do not usually develop anti-HLA-antibodies specific to antigens that belong to their own CREGphenotype.3,4 This observation could support the hypothesis that the acceptable mismatched antigens in sensitized patients belong to the same CREG with the recipients CREG-phenotype. To analyze the previous possibility, we evaluated the specificity of anti-HLA-antibodies directed against intraCREG-mismatches (intra-CREG-MMs) in alloimmunized patients.
rized as intra-CREGs-MMs and other-CREGs-MMs. The same classification was used for the specificities of the detected anti-HLA-antibodies. Analysis of anti-HLA specificity. Antibody specificity of the AHG-CDC data was based on 2 3 2 table analysis using chi square statistics to determine significant correlations between serum reactivity patterns and the presence of specific markers in the cell panel. In addition, the SOFTSTAT program analysis data was used for the definition of the antibody specificity detected by ELISA. The SOFTSTAT program performs analysis of antibody specificity, by the Fisher Exact Test. RESULTS
Forty-nine patients (49/50, 98%) had received grafts with at least one class I HLA-mismatched-antigen (Table 1) and in 63.2% (31/49) were found intra-CREGs-MMs. Thirty-six patients developed anti-HLA-antibodies. Antibodies with intra-CREGs-specificity were detected in 29% (9/31) of the patients (Table 1). All these anti-HLA-antibodies were donor specific. Antibodies with other-CREGs-MMs were detected in 37/42 (88%) cases. Twenty-six (26/37, 70%) of these anti-HLA-antibodies were donor specific (Table 1). In Table 2 is shown the HLA-specificity of the anti-HLAantibodies in the screening panel, which is compared with the intra-CREG-MMs in nine patients.
MATERIALS AND METHODS The study group consisted of 50 patients (33 males and 17 females), mean age 46 6 8 years. Thirty one (31) patients had a previous renal graft loss, and nineteen (19) patients were renal transplant recipients with biopsy proven chronic rejection (CR). Three hundred ten (310) serum samples collected prior transplantation, at various times posttransplantation, and after the graft loss in the 31 patients, were tested for anti-HLA-antibodies. The %PRA and the specificity of anti-HLA-antibodies were studied by complement dependent cytotoxicity (CDC) and Elisa technique. Cytotoxicity testing for class I anti-HLA-Abs was performed on T lymphocytes from 60 HLA-phenotyped donors using the anti-human globulin (AHG-CDC) augmented modification of the CDC technique.5,6 The PRA-STAT test (SangStat Medical Corp.) was performed according to the manufacturer’s instructions.7
HLA-phenotype. All recipients and donors were typed for class I HLA-antigens by a standard lymphocytotoxicity technique. The mismatched HLA-antigens were catego© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
DISCUSSION
It is generally accepted that humoral immunity plays a prominent role in graft rejection. This became apparent when preformed recipient antidonor anti-HLA-antibodies were shown to cause hyperacute rejection.8 In contrast, the data concerning the relevance of anti-HLA-antibodies appearing posttransplant are more controversial. Some studies have shown that the production of posttransplant antiFrom the Department of Immunology and National Tissue Typing Center, General Hospital of Athens “Georgios Gennimatas,” Department of Nephrology, and Transplant Unit, Laikon General Hospital, Athens, Greece. Address reprint requests to Andreas Papassavas, PhD, Department of Immunology and National Tissue Typing Center, General Hospital of Athens “Georgios Gennimatas,” Mesogion 154 Ave, 11527, Athens, Greece. E-mail: [email protected]. 0041-1345/99/$–see front matter PII S0041-1345(98)01751-5 757
Transplantation Proceedings, 31, 757–759 (1999)
758
PAPASSAVAS, INIOTAKI–THEODORAKI, BOLETIS ET AL
Table 1. Evaluation of the Donor CREGs-Mismatches and the Donor-Specific Class I Anti-HLA-Antibodies in 49 Renal Transplant Recipients
Graft mismatches 1. Only intra-CREGs-mismatches Anti-HLA-antibodies formation Donor specific Without anti-HLA-antibodies 2. Other and intra-CREGs-mismatches* Anti-HLA-antibodies formation Donor specific intra-CREGs-MMs Donor specific other-CREGs-MMs Non donor specific other-CREGsMMs With specificity against other-non MMs-CREGs Without anti-HLA-antibodies
n
%
49 7
98 14.29
4 3 42
57.14 42.86 85.71
5 26 3
11.9 61.9 7.14
8 10
19 23.8
*Twenty-four patients had both intra- and other-CREG-MMs and 18 patients had only other-CREG-MMs.
HLA-antibodies is associated with poor graft outcome,9,10 and anti-HLA-antibodies have been eluted from rejected kidney allografts.11,12 Moreover, it has been established that preformed IgG anti-HLA-antibodies specific for donor HLA-antigens may result in accelerated graft failure.8 However, other data are contradictory, and the relevance of posttransplant anti-HLA-antibodies remains to be defined.13 It is obvious that the detection of PRA after
transplantation can act only as an indicator of anti-donor reactivity in the recipient and that the detected specificity of the anti-HLA-antibodies, rather than isotype, is of key importance.14,15 The results of this study support the observation that graft recipients perform antibodies against their own CREG mismatched antigens in a smaller degree, compared to other CREG mismatched antigens (29% vs 70%). The observation that all these antibodies were donor specific, and possibly directed against private epitopes of the donor HLA-mismatched antigens, is of much interest. This observation could be explained by the hypothesis that HLAantigens that belong to the same CREG carry both public and private epitopes. Alloreactivity or non–reactivity of recipients immune system depends on which epitope is presented to the alloreactive T cells. The immunogenic private epitopes are presented to specific alloreactive T cells with consequence lymphokine secretion providing help for allontibody production. Public and possibly nonimmunogenic epitopes presented to recipients T cells produce tolerance rather than reactivity. Consequently, a matching strategy based on the detection of acceptable mismatches, which belong to the recipient own CREG-phenotype could be used in order to increase the pool of the most suitable donors,16 although this strategy seems not to be adequate enough to replace the conventional HLA-matching. Thus, the epitope analysis of the anti-HLA-antibodies with intra-
Table 2. HLA-Specificity Analysis of the Anti-HLA-Antibodies in the Screening Panel and Comparison With the Intra-CREGs-MMs Patients (n 5 50)
Patient HLA-phenotype
1.
A2, A11, B7, B18, DR8, DR11
2C, 1C, 7C, 8C, 6C
2.
A23, A28, B44, B51, DR2, DR5
1C, 2C, 12C, 5C, 4C
3.
A24, A28, B18, Bx, DR11, DR16
1C, 2C, 8C, 6C
4.
A24, Ax, B13, B35, DR4, DR7
1C, 7C, 5C, 4C, 6C
5.
A2, A11, B44, B60, DR1, DR5
2C, 1C, 12C, 7C, 4C, 6C
6.
A24, A28, B18, B39, DR2, DR5
1C, 2C, 8C, 6C
7.
A2, A30, B13, B51, DR10, DR11 A1, A26, B40, B51, DR5, DR14
2C, 1C, 7C, 5C, 4C 1C, 10C, 7C, 5C, 6C, 4C
A26, A28, B35, B51, DR1, DR13
10C, 2C, 5C, 4C, 6C
8.
9.
Patient CREGs
Donor HLA-phenotype
A24, A29, B7, B8, DR10, DR11 A1, Ax, B52, B57, DR2, DR9 A2, A23, B18, B14, DR11, DR13 A2, A3, B13, B35, DR2, DR7 A2, A3, B7, B44, DR5, DRx A1, A2, B18, B57, DR2, DR5 A1, A2, B8, B51, DR2, DR10 A1, A23, B51, Bx, DR5, DRx A2, B18, B57, DR5, DR7
Intra CREG-MMs
Intra CREGs
1C, 7C, 8C, 6C
A24
1C
A24
1C
1C, 5C, 21C, 4C
A1
1C
A1
1C, 21C
2C, 1C, 8C, 6C
A2, A23
2C, 1C
A2
2C
2C, 1C, 7C, 5C, 4C, 6C
A3
1C
A3
1C
2C, 1C, 7C, 12C, 6C, 4C 1C, 2C, 8C, 21C, 6C, 4C 1C, 2C, 8C, 5C, 6C, 4C
A3, B7
1C, 7C
B7
7C
A1, A2
1C, 2C
A1
1C
A1
1C
A1
1C
1C, 5C, 4C
A23
1C
A23
1C
2C, 8C, 21C, 4C, 6C
A2
2C
A2
2C
Donor CREGs
Anti-HLA Specificity
Anti-HLA CREGs
DEVELOPMENT OF ANTI-HLA-ANTIBODIES
CREG-specificity could be useful for the detection of the non-acceptable intra-CREG-antigens. REFERENCES 1. Rodey GE, Fuller TC: CRC Ctic Rev Immunol 7:229, 1987 2. Fuller TC: Clin Lab Med 11:551, 1991 3. Rodey GE, Neylan JF, Whelchel JD, et al: Hum Immunol 39:272, 1994 4. Delmonico FL, Fuller A, Cosimi AB, et al: Transplantation 36:629, 1983 5. Johnson AH, Rossen RD, and Butler WT: Tissue Antigens 2:215, 1972 6. Hopkins KA: In Zachary AA, Terisi GA (eds): ASHI Laboratory Manual, 2nd ed. 1990, p. 195 7. Zachary AA, Griffin J, Lucas DP, et al: Transplantation 60:1600, 1995
759 8. Kissmeyer-Nielsen F, Olsen S, Petersen VP, et al: Lancet 1:662, 1966 9. Nunez G, McPhaul JR, Stastny P: Transplantation 35:446, 1983 10. London RE, Stelzer GT, Ferrone S: Transplantation 38:493, 1984 11. Vogeler U, Doxiadis I, Eigler FW, et al: Transplant Proc 17:2630, 1985 12. Martin S, Dyer P, Mallick NP, et al: Transplantation 44:50, 1987 13. Ting A, Morris PI: Transplantation 28:13, 1979 14. Worthington JE, Thomas AA, Dyer PA, et al: Transplantation 65:121, 1998 15. Rodey GE, Revels K, and Fuller TC: Transplantation 63: 885, 1997 16. Papassavas AC, Iniotaki-Theodoraki A, Boletis J, et al: Hum Immunol 59:587, 1998