- Email: [email protected]
Alloantibodies and outcomes of deceased donor kidney allografts
Human Immunology 70 (2009) 651– 654
Contents lists available at ScienceDirect
Alloantibodies and outcomes of deceased donor kidney allografts P. Cinti, R. Pretagostini, Q. Lai*, M.L. Tamburro, M. Rossi, L. Poli, P. Berloco Chirurgia Generale E Trapianti d’Organo, La Sapienza UniversitÞ di Roma, Umberto I Policlinico di Roma, Rome, Italy
A R T I C L E
I N F O
Article history: Received 1 April 2009 Accepted 9 June 2009 Available online 12 June 2009
Keywords: Alloantibodies Kidney transplantation
A B S T R A C T
Analysis of the anti-HLA antibody status of 100 recipients of kidneys from deceased donors demonstrated that presensitization and the development of alloantibodies after transplantation are associated with the development of antibody mediated as well as cellular rejection. This finding indicates that the humoral arm of the immune response is also involved in cell-mediated rejection and/or that there may be a continuum between these two forms of rejection. Most episodes of rejection were successfully reversed in our population, as shown by the overall 3-year actuarial survival of 98% in nonsensitized and 91% in sensitized recipients, emphasizing the importance of comprehensive antibody studies. 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The role of anti– human leukocyte antibody (HLA) antibodies as mediators of allograft rejection has long been recognized. Allosensitization is generally caused by exposure of the recipient to mismatched HLA antigens from transfusions, previous transplants or pregnancies. The detection of alloantibodies relies on different methods such as complement-dependent cytotoxicity (CDC), flow cytometry (FC), and solid phase assay (SPA).These methods can be used for pre- and post-transplantation monitoring of the frequency and specificity of anti-HLA antibodies as well for crossmatching (CX) the recipients’ sera with T and B lymphocytes from potential donors. Recent studies have shown that FC and SPA have an increased sensitivity compared with CDC yet frequently detect alloantibodies that are not harmful to the graft, either because they do not fix complement or because their titer and affinity are very low [1–7]. In our institution, the birthplace of kidney transplantation in Italy (1966), an effort has been made over the last years to integrate new technology, such as FC and SPA, into the standard protocol for alloantibody detection, which is based on CDC. We now report the actuarial 3-year graft survival, the incidence, and type of rejection episodes and their relationship with the patients’ anti-HLA antibody status presented in a series of 100 consecutive transplants of kidneys from deceased donors on the results. 2. Subjects and methods 2.1. Patients The patient population consisted of 92 recipients of primary and eight of secondary kidney allografts. Anti-HLA class I and class II * Corresponding author. E-mail address: [email protected] (L. Quirino).
antibodies were found before kidney transplantation in the circulation of 9% and 10% of the patients, respectively using CDC and SPA screening procedures for the identification of presensitized transplant candidates. 2.2. Antibody studies Sequential samples of pretransplantation sera were collected at 3-month intervals from all patients. Post-transplantation sera were obtained whenever antibody-mediated rejection (AMR) was suspected. Panel reactive antibodies (PRA) were studied by CDC and their specificity was determined by SPA according to standard procedures. Donor-specific crossmatching was performed by CDC-XM using magnetically sorted T and B cells from the donor’s spleen and by FC using mouse anti-human CD3 and CD19 antibodies to define target T and B cells, respectively. Flow cytometry crossmatch (FC-XM)– positive sera were tested for antibody specificity using LABScreen single antigen beads (One Lambda, Los Angeles, CA). 2.3. Treatment All patients received basilimax as an induction agent and triple therapy with calcineurine inhibitors, microphenolic acid, and steroids for maintenance of immunosuppression. Biopsyproven acute cellular rejection episodes were treated with steroids (1g/day) for 3 days. Patients with biopsy-proven AMR who showed C4D deposition in the graft and donor-specific antibodies (DSA) in the circulation received steroid boluses for 3 days, plasmapheresis with immunoabsorbtion for 3 consecutive days and then on alternate days until the crossmatch with the donor became negative, and DSA were no longer detectable in the circulation. The average number of immunoabsorption sessions was 6.5 ⫾ 1.4 (range, 5–9 days). After depletion of DSA, IVIG (0.4 mg/kg/day) was administered for 3 days.
0198-8859/09/$32.00 - see front matter 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2009.06.008
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P. Cinti et al. / Human Immunology 70 (2009) 651– 654
2.4. Pathologic examination Biopsies were performed for delayed graft function or graft dysfunction, defined as a rise in serum creatinine, BUN, and/or proteinuria. Rejection was diagnosed by immunohistochemical staining of graft biopsy samples according to Banff criteria. AMR was considered to be present when there was morphologic evidence of tissue injury, serologic evidence of DSA, and diffuse C4d staining. Acute cellular rejection (ACR) was defined by tubulitis, interstitial inflammation, and/or arterial inflammation with T-cell and macrophage infiltrates. 2.5. Statistical analysis Actuarial kidney allograft survival was estimated by the KaplanMeier method and compared using the log-rank test. Categorical variables were compared with Fisher exact test or the 2 test. All statistical analyses were performed with SPSS software, version 14.0. (SPSS, Inc., Chicago, IL). 3. Results 3.1. Allograft survival in patients with or without anti-HLA alloantibodies before transplantation Although a negative crossmatch of patient’s sera with the donor lymphocytes indicates the absence of DSA, it does not exclude the possibility that allosensitization against irrelevant HLA antigens increases the risk of rejection. Table 1 shows the relationship between the presence or absence of antibodies to HLA class I and/or II and development of ACR. Rejection was positively associated with anti-HLA class I (p ⫽ 0.03) but not class II antibodies. Of the 16 cases of ACR, four occurred in patients with anti-HLA alloantibodies. Because four of the nine patients who had anti– class I antibodies developed ACR, it appears that such antibodies may opsonize the graft due to crossreactivity with certain epitopes of donor HLA antigens. In the absence of inflammation, the very low level of expression of HLA class II antigens on graft endothelial cells may explain the finding that anti-HLA class II antibodies have a lesser impact. Analysis of the relationship between presensitization, reflected in non-DSA alloantibodies, and development of AMR showed a highly significant association between the presence in pretransplantation sera of antibodies to either class I (p ⫽ 0.002) (Table 1) or class II antigens (p ⫽ 0.003) and development of AMR, indicating that allosensitized patients are more likely to develop DSA after
Table 1 ACR and AMR in recipients with and without anti-HLA antibodies before transplantation ACR ⫹ Antibodies to Both HLA class I and II antigens Class I antigens only Class II antigens only No antibodies
Antibodies to HLA to class I antigens No class I antibodies
Antibodies to HLA to class II antigens No class II antibodies
3
AMR ⫹
⫺
3
6
1 2 0 4 12 75 16 84 p ⫽ 0.07
3
6
3 4 87 100
0 3 0 4 1 86 4 96 p ⬍ 0.0001
3 4 87 100
4 5 12 79 16 84 p ⫽ 0.03
9 87 100
3 6 1 90 4 96 p ⫽ 0.002
9 81 100
3 13 16 NS
10 90 100
3 7 1 89 4 96 p ⫽ 0.003
10 90 100
7 77 84
3
⫺
transplantation than are patients with no history of alloantibodies. Of note, AMR also occurred in one patient with no pretransplantation anti-HLA alloantibodies, suggesting that the AMR could have been caused by antibodies to antigens other than HLA. The impact of presensitization on allograft survival is shown in Fig. 1. At 3 years the actuarial graft survival in patients with no history of anti-HLA antibodies (n ⫽ 87) was 98%, compared with 91% in patients with antibodies to HLA class I and/or II antigens (n ⫽ 13). Analysis of allograft survival in patients who had only anti-HLA class I (n ⫽ 9) or class II (n ⫽ 10) antibodies shows a further reduction of long term graft function to 86%. However, because of the relatively low number of patients who lost the graft because of cellular or humoral rejection, the difference between the groups was not statistically significant. Overall the actuarial graft survival in recipients of primary grafts (n ⫽ 92) and secondary grafts (n ⫽ 8) was 96% and 100%, respectively (p ⫽ NS). 3.2. Allograft survival in recipients with or without anti-HLA alloantibodies post-transplantation Screening of anti-HLA antibodies in post-transplantation sera revealed their development in a fraction of the patient population. There was a significant correlation (p ⫽ 0.01) between the development of ACR (n ⫽ 16) and that of antibodies to HLA class I (n ⫽ 3), class II (n ⫽ 1) or both class I and II antigens (n ⫽ 4). This association was stronger with anti-HLA class I (p ⫽ 0.007) than with class II (p ⫽ 0.04), consistent with the notion that the wider distribution and higher level of expression of class I antigens renders them more vulnerable to cross-reactive antibodies that may opsonize graft tissues, triggering ACR (Table 2). However, analysis of the relationship between AMR and presence of alloantibodies in sera obtained after transplantation showed a highly significant association when both anti– class I and II antibodies were considered together (p ⫽ 0.0002), yet less significant p values when only class I (p ⫽ 0.03) or class II (p ⫽ NS) were considered (Table 2). These data imply that presensitization to class II antigens does not result in greater risk for AMR unless anti– class I antibodies are also present, consistent with other authors’ findings [7–9]. 3.3. Post-transplantation crossmatches and rejection Direct crossmatching of sera obtained from the recipient with donor lymphocytes was performed in all cases using both FC and CDC techniques when rejection was suspected. By FC-XM we found a highly significant correlation between DSA and ACR (p ⫽ 0.003). Of 16 patients with ACR, eight displayed DSA at the time of rejection, indicating that although these episodes were typically “cellular”; that is, they responded to steroid treatment, required no immunoabsorbtion, and showed no C4d deposition on biopsy, a strong humoral component was still involved. FC-XM was positive in all four cases of AMR, yet also yielded positive results in four cases without AMR (p ⫽ 0.003). The specificity of the FC-XM-detected antibodies for donor HLA antigens was confirmed by SPA using single-antigen– coated beads. The finding that FC-XM was false positive in four cases (without AMR) indicates that not all alloantibodies detected by this method are detrimental to the group (Table 3). By CDC-XM there was also a positive association between the presence of DSA and ACR, as the crossmatch was positive in six of 15 ACR cases (p ⫽ 0.02). Similarly, CDC-XM was positive in three of four AMR cases and in three cases without AMR (p ⫽ 0.03) (Table 3). 4. Discussion The current study re-emphasizes the well-accepted notion that detection and monitoring of DSA is crucial to the outcome of kidney allografts. Newly developed strategies for immunologic evaluation
P. Cinti et al. / Human Immunology 70 (2009) 651– 654
653
Fig. 1. (A) Actuarial survival of renal allograft in recipients with and without alloantibodies. (B) Actuarial survival of renal allograft in patients with and without presensitization to HLA– class I antigens. (C) actuarial Survival of renal allograft in patients with and without presensitization to HLA– class II antigens.
of transplant candidates include CDC, SPA and FC techniques for antibody screening and donor-recipient crossmatching. Despite extensive controversy regarding the specificity and sensitivity of each of these procedures, there is a consensus on the importance about using them in concert to optimize patients’ chances to undergo transplantation successfully [1–7]. The results that we obtained in this cohort of 100 recipients of kidneys from deceased donors testifies to this effect, as the overall the actuarial survival at 3 years was 96% for primary and 100% for secondary transplants, dspite a 13% presensitization rate. A rather new and surprising finding in this study was the positive relationship between the presence of anti-HLA class I antibod-
ies in patients’ circulation (18) before and/or after transplantation and ACR. Although the association with AMR was only to be expected, the positive correlation with cellular rejection calls for an explanation. It has been known for more than a decade that cellular rejection is mediated by T cells activated via the direct and indirect pathways of allorecognition [9 –11]. We previously demonstrated an increased frequency of allopeptide reactive T cells in the circulation of patients undergoing cellular or chronic, humoral rejection [12– 14]. We postulated that allopeptide reactive T cells differentiate upon recognition of HLA antigens from the injured graft on self APC that have processed donor cells killed via the direct pathway. Such
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P. Cinti et al. / Human Immunology 70 (2009) 651– 654
Table 2 ACR and AMR in recipients with and without anti-HLA antibodies post-transplantation ACR ⫹ Antibodies to Both HLA class I and II antigens Class I antigens only Class II antigens only No antibodies
Antibodies to HLA to class I antigens No class I antibodies
Antibodies to HLA to class II antigens No class II antibodies
4
AMR ⫹
⫺
0
4
3 0 1 0 8 16 16 16 p ⫽ 0.01
1
⫺
3
4
3 1 24 32
2 1 1 0 0 24 4 28 p ⫽ 0.0002
3 1 24 32
7 0 9 16 16 16 p ⫽ 0.007
7 25 32
3 4 1 24 4 28 p ⫽ 0.03
7 25 32
5 0 11 16 16 16 p ⫽ 0.04
5 27 32
2 3 2 25 4 28 p ⫽ NS
5 27 32
allopeptide reactive T cells produce cytokines that stimulate the maturation of other immunocytes, including alloantibody producing B cells, natural killer cells, macrophages, and dendritic cells. It follows from this scenario that factors facilitating the onset of acute rejection (mediated by T cells with direct allorecognition ability), Table 3 ACR and AMR in patients developing FC-XM and CDC-XM detectable DSA post-transplantation ACR
Post-transplant FC-XM⫹ Negative FC-XM
Post-transplant FCXM⫹ Negative FC-XM
Post-transplant CDC-XM⫹ Negative CDC-XM
Post-transplant CDC-XM⫹ Negative CDC-XM
⫹
⫺
8 8 16 p ⫽ 0.003 AMR
0 13 13
8 21 29
⫹ 4 0 4 p ⫽ 0.003 ACR
⫺ 4 21 25
8 21 29
⫹ 6 9 15 p ⫽ 0.02 AMR
⫺ 0 11 11
6 20 26
⫹ 3 1 4 p ⫽ 0.03
⫺ 3 19 22
6 20 26
such as cross-reactive alloantibodies that opsonize graft cells, may promote inflammation and ACR. It is obvious, however, that early diagnosis and treatment of ACR restores quiescence in most patients, probably by allowing chronic stimulation of noncytolytic CD8⫹ T cells that are MHC class I allorestricted and acquire suppressor function [19]. It is equally important to notice that, in our experience, AMR is a reversible phenomenon when diagnosed and treated in a timely and efficient manner [15–17]. Taken together, our data emphasizes the importance of antibody studies for management of patients receiving organ allografts. Acknowledgements The statistical analysis was supported by the Interuniversitary Organ Transplantation Consortium, Rome, Italy. References [1] Singh N, Pirsch J, Samaniego M. Antibody-mediated rejection: Treatment alternatives and outcomes. Transplant Rev 2009;23:34– 46. [2] Jackson AM, Zachary AA. The problem of transplanting the sensitized patient: Whose problem is it? Front Biosci 2008;13:1396 – 412. [3] Jordan SC, Pescovitz MD. Presensitization: The problem and its management. Clin J Am Soc Nephrol 2006;1:421–32. [4] Zeevi A, Girnita A, Duquesnoy R. HLA antibody analysis: Sensitivity, specificity, and clinical significance in solid organ transplantation. Immunol Res 2006;36: 255– 64. [5] Tinckam KJ, Chandraker A. Mechanisms and role of HLA and non-HLA alloantibodies. Clin J Am Soc Nephrol 2006;1:404 –14. [6] Vasilescu ER, Ho EK, Colovai AI, Vlad G, Foca-Rodi A, Markowitz GS, et al. Alloantibodies and the outcome of cadaver kidney allografts. Hum Immunol 2006;67:597– 604. [7] Ho EK, Vasilescu ER, Colovai AI, Stokes MB, Hallar M, Markowitz GS, et al. Sensitivity, specificity and clinical relevance of different cross-matching assays in deceased-donor renal transplantation. Transpl Immunol 2008;20:61–7. [8] Su¨sal C, Opelz G. Kidney graft failure and presensitization against HLA class I and class II antigens. Transplantation 2002;73:1269 –73. [9] Su¨sal C, Opelz G. Good kidney transplant outcome in recipients with presensitization against HLA class II but not HLA class I. Hum Immunol 2004;65:810 – 6. [10] Liu Z, Colovai AI, Tugulea S, Reed EF, Fisher PE, Mancini D, et al. Indirect recognition of donor HLA-DR peptides in organ allograft rejection. J Clin Invest 1996;98:1150 –7. [11] Ciubotariu R, Liu Z, Colovai AI, Ho E, Itescu S, Ravalli S, et al. Persistent allopeptide reactivity and epitope spreading in chronic rejection of organ allografts. J Clin Invest 1989;101:398 – 405. [12] Renna-Molajoni E, Cinti P, Orlandini AM, Molajoni J, Cocciolo PL, Evangelista B, et al. Contribution of the direct and indirect allorecognition pathway to the rejection of liver allografts. Transplant Proc 1998;30:2138 –9. [13] Renna-Molajoni E, Cinti P, Evangelista B, Orlandini AM, Molajoni J, Cocciolo PL, et al. Role of the indirect recognition pathway in the development of chronic liver allograft rejection. Transplant Proc 1998;30:2140 –1. [14] Renna-Molajoni E, Cinti P, Elia L, Orlandini AM, Cocciolo P, Molajoni J, et al. Mechanism of liver allograft rejection: Indirect allorecognition. Transplant Proc 1999;31:409 –10. [15] Ciubotariu R, Vasilescu R, Ho E, Cinti P, Cancedda C, Poli L, et al. Detection of T suppressor cells in patients with organ allografts. Hum Immunol 2001;62:15– 20. [16] Molajoni ER, Cinti P, Ho E, Evangelista B, Lonardo MT, Rossi M, et al. Allospecific T-suppressor cells in liver transplantation. Transplant Proc 2001;33:1381–3. [17] Renna Molajoni E, Cinti P, Ho E, Evangelista B, Peritore D, Poli L, et al. Allospecific human T suppressor cells in kidney transplantation. Transplant Proc 2001;33:1129 –30. [18] Su¨sal C, D×hler B, Sadeghi M, Ovens J, Opelz G. HLA antibodies and the occurrence of early adverse events in the modern era of transplantation: a collaborative transplant study report. Transplantation 2009;87(9):2367–71. [19] Vlad G, Suciu-Foca N. Resurgence or emergence of CD8⫹ Ts. Hum Immunol 2008;69:679 – 80.
Contents lists available at ScienceDirect
Alloantibodies and outcomes of deceased donor kidney allografts P. Cinti, R. Pretagostini, Q. Lai*, M.L. Tamburro, M. Rossi, L. Poli, P. Berloco Chirurgia Generale E Trapianti d’Organo, La Sapienza UniversitÞ di Roma, Umberto I Policlinico di Roma, Rome, Italy
A R T I C L E
I N F O
Article history: Received 1 April 2009 Accepted 9 June 2009 Available online 12 June 2009
Keywords: Alloantibodies Kidney transplantation
A B S T R A C T
Analysis of the anti-HLA antibody status of 100 recipients of kidneys from deceased donors demonstrated that presensitization and the development of alloantibodies after transplantation are associated with the development of antibody mediated as well as cellular rejection. This finding indicates that the humoral arm of the immune response is also involved in cell-mediated rejection and/or that there may be a continuum between these two forms of rejection. Most episodes of rejection were successfully reversed in our population, as shown by the overall 3-year actuarial survival of 98% in nonsensitized and 91% in sensitized recipients, emphasizing the importance of comprehensive antibody studies. 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The role of anti– human leukocyte antibody (HLA) antibodies as mediators of allograft rejection has long been recognized. Allosensitization is generally caused by exposure of the recipient to mismatched HLA antigens from transfusions, previous transplants or pregnancies. The detection of alloantibodies relies on different methods such as complement-dependent cytotoxicity (CDC), flow cytometry (FC), and solid phase assay (SPA).These methods can be used for pre- and post-transplantation monitoring of the frequency and specificity of anti-HLA antibodies as well for crossmatching (CX) the recipients’ sera with T and B lymphocytes from potential donors. Recent studies have shown that FC and SPA have an increased sensitivity compared with CDC yet frequently detect alloantibodies that are not harmful to the graft, either because they do not fix complement or because their titer and affinity are very low [1–7]. In our institution, the birthplace of kidney transplantation in Italy (1966), an effort has been made over the last years to integrate new technology, such as FC and SPA, into the standard protocol for alloantibody detection, which is based on CDC. We now report the actuarial 3-year graft survival, the incidence, and type of rejection episodes and their relationship with the patients’ anti-HLA antibody status presented in a series of 100 consecutive transplants of kidneys from deceased donors on the results. 2. Subjects and methods 2.1. Patients The patient population consisted of 92 recipients of primary and eight of secondary kidney allografts. Anti-HLA class I and class II * Corresponding author. E-mail address: [email protected] (L. Quirino).
antibodies were found before kidney transplantation in the circulation of 9% and 10% of the patients, respectively using CDC and SPA screening procedures for the identification of presensitized transplant candidates. 2.2. Antibody studies Sequential samples of pretransplantation sera were collected at 3-month intervals from all patients. Post-transplantation sera were obtained whenever antibody-mediated rejection (AMR) was suspected. Panel reactive antibodies (PRA) were studied by CDC and their specificity was determined by SPA according to standard procedures. Donor-specific crossmatching was performed by CDC-XM using magnetically sorted T and B cells from the donor’s spleen and by FC using mouse anti-human CD3 and CD19 antibodies to define target T and B cells, respectively. Flow cytometry crossmatch (FC-XM)– positive sera were tested for antibody specificity using LABScreen single antigen beads (One Lambda, Los Angeles, CA). 2.3. Treatment All patients received basilimax as an induction agent and triple therapy with calcineurine inhibitors, microphenolic acid, and steroids for maintenance of immunosuppression. Biopsyproven acute cellular rejection episodes were treated with steroids (1g/day) for 3 days. Patients with biopsy-proven AMR who showed C4D deposition in the graft and donor-specific antibodies (DSA) in the circulation received steroid boluses for 3 days, plasmapheresis with immunoabsorbtion for 3 consecutive days and then on alternate days until the crossmatch with the donor became negative, and DSA were no longer detectable in the circulation. The average number of immunoabsorption sessions was 6.5 ⫾ 1.4 (range, 5–9 days). After depletion of DSA, IVIG (0.4 mg/kg/day) was administered for 3 days.
0198-8859/09/$32.00 - see front matter 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2009.06.008
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P. Cinti et al. / Human Immunology 70 (2009) 651– 654
2.4. Pathologic examination Biopsies were performed for delayed graft function or graft dysfunction, defined as a rise in serum creatinine, BUN, and/or proteinuria. Rejection was diagnosed by immunohistochemical staining of graft biopsy samples according to Banff criteria. AMR was considered to be present when there was morphologic evidence of tissue injury, serologic evidence of DSA, and diffuse C4d staining. Acute cellular rejection (ACR) was defined by tubulitis, interstitial inflammation, and/or arterial inflammation with T-cell and macrophage infiltrates. 2.5. Statistical analysis Actuarial kidney allograft survival was estimated by the KaplanMeier method and compared using the log-rank test. Categorical variables were compared with Fisher exact test or the 2 test. All statistical analyses were performed with SPSS software, version 14.0. (SPSS, Inc., Chicago, IL). 3. Results 3.1. Allograft survival in patients with or without anti-HLA alloantibodies before transplantation Although a negative crossmatch of patient’s sera with the donor lymphocytes indicates the absence of DSA, it does not exclude the possibility that allosensitization against irrelevant HLA antigens increases the risk of rejection. Table 1 shows the relationship between the presence or absence of antibodies to HLA class I and/or II and development of ACR. Rejection was positively associated with anti-HLA class I (p ⫽ 0.03) but not class II antibodies. Of the 16 cases of ACR, four occurred in patients with anti-HLA alloantibodies. Because four of the nine patients who had anti– class I antibodies developed ACR, it appears that such antibodies may opsonize the graft due to crossreactivity with certain epitopes of donor HLA antigens. In the absence of inflammation, the very low level of expression of HLA class II antigens on graft endothelial cells may explain the finding that anti-HLA class II antibodies have a lesser impact. Analysis of the relationship between presensitization, reflected in non-DSA alloantibodies, and development of AMR showed a highly significant association between the presence in pretransplantation sera of antibodies to either class I (p ⫽ 0.002) (Table 1) or class II antigens (p ⫽ 0.003) and development of AMR, indicating that allosensitized patients are more likely to develop DSA after
Table 1 ACR and AMR in recipients with and without anti-HLA antibodies before transplantation ACR ⫹ Antibodies to Both HLA class I and II antigens Class I antigens only Class II antigens only No antibodies
Antibodies to HLA to class I antigens No class I antibodies
Antibodies to HLA to class II antigens No class II antibodies
3
AMR ⫹
⫺
3
6
1 2 0 4 12 75 16 84 p ⫽ 0.07
3
6
3 4 87 100
0 3 0 4 1 86 4 96 p ⬍ 0.0001
3 4 87 100
4 5 12 79 16 84 p ⫽ 0.03
9 87 100
3 6 1 90 4 96 p ⫽ 0.002
9 81 100
3 13 16 NS
10 90 100
3 7 1 89 4 96 p ⫽ 0.003
10 90 100
7 77 84
3
⫺
transplantation than are patients with no history of alloantibodies. Of note, AMR also occurred in one patient with no pretransplantation anti-HLA alloantibodies, suggesting that the AMR could have been caused by antibodies to antigens other than HLA. The impact of presensitization on allograft survival is shown in Fig. 1. At 3 years the actuarial graft survival in patients with no history of anti-HLA antibodies (n ⫽ 87) was 98%, compared with 91% in patients with antibodies to HLA class I and/or II antigens (n ⫽ 13). Analysis of allograft survival in patients who had only anti-HLA class I (n ⫽ 9) or class II (n ⫽ 10) antibodies shows a further reduction of long term graft function to 86%. However, because of the relatively low number of patients who lost the graft because of cellular or humoral rejection, the difference between the groups was not statistically significant. Overall the actuarial graft survival in recipients of primary grafts (n ⫽ 92) and secondary grafts (n ⫽ 8) was 96% and 100%, respectively (p ⫽ NS). 3.2. Allograft survival in recipients with or without anti-HLA alloantibodies post-transplantation Screening of anti-HLA antibodies in post-transplantation sera revealed their development in a fraction of the patient population. There was a significant correlation (p ⫽ 0.01) between the development of ACR (n ⫽ 16) and that of antibodies to HLA class I (n ⫽ 3), class II (n ⫽ 1) or both class I and II antigens (n ⫽ 4). This association was stronger with anti-HLA class I (p ⫽ 0.007) than with class II (p ⫽ 0.04), consistent with the notion that the wider distribution and higher level of expression of class I antigens renders them more vulnerable to cross-reactive antibodies that may opsonize graft tissues, triggering ACR (Table 2). However, analysis of the relationship between AMR and presence of alloantibodies in sera obtained after transplantation showed a highly significant association when both anti– class I and II antibodies were considered together (p ⫽ 0.0002), yet less significant p values when only class I (p ⫽ 0.03) or class II (p ⫽ NS) were considered (Table 2). These data imply that presensitization to class II antigens does not result in greater risk for AMR unless anti– class I antibodies are also present, consistent with other authors’ findings [7–9]. 3.3. Post-transplantation crossmatches and rejection Direct crossmatching of sera obtained from the recipient with donor lymphocytes was performed in all cases using both FC and CDC techniques when rejection was suspected. By FC-XM we found a highly significant correlation between DSA and ACR (p ⫽ 0.003). Of 16 patients with ACR, eight displayed DSA at the time of rejection, indicating that although these episodes were typically “cellular”; that is, they responded to steroid treatment, required no immunoabsorbtion, and showed no C4d deposition on biopsy, a strong humoral component was still involved. FC-XM was positive in all four cases of AMR, yet also yielded positive results in four cases without AMR (p ⫽ 0.003). The specificity of the FC-XM-detected antibodies for donor HLA antigens was confirmed by SPA using single-antigen– coated beads. The finding that FC-XM was false positive in four cases (without AMR) indicates that not all alloantibodies detected by this method are detrimental to the group (Table 3). By CDC-XM there was also a positive association between the presence of DSA and ACR, as the crossmatch was positive in six of 15 ACR cases (p ⫽ 0.02). Similarly, CDC-XM was positive in three of four AMR cases and in three cases without AMR (p ⫽ 0.03) (Table 3). 4. Discussion The current study re-emphasizes the well-accepted notion that detection and monitoring of DSA is crucial to the outcome of kidney allografts. Newly developed strategies for immunologic evaluation
P. Cinti et al. / Human Immunology 70 (2009) 651– 654
653
Fig. 1. (A) Actuarial survival of renal allograft in recipients with and without alloantibodies. (B) Actuarial survival of renal allograft in patients with and without presensitization to HLA– class I antigens. (C) actuarial Survival of renal allograft in patients with and without presensitization to HLA– class II antigens.
of transplant candidates include CDC, SPA and FC techniques for antibody screening and donor-recipient crossmatching. Despite extensive controversy regarding the specificity and sensitivity of each of these procedures, there is a consensus on the importance about using them in concert to optimize patients’ chances to undergo transplantation successfully [1–7]. The results that we obtained in this cohort of 100 recipients of kidneys from deceased donors testifies to this effect, as the overall the actuarial survival at 3 years was 96% for primary and 100% for secondary transplants, dspite a 13% presensitization rate. A rather new and surprising finding in this study was the positive relationship between the presence of anti-HLA class I antibod-
ies in patients’ circulation (18) before and/or after transplantation and ACR. Although the association with AMR was only to be expected, the positive correlation with cellular rejection calls for an explanation. It has been known for more than a decade that cellular rejection is mediated by T cells activated via the direct and indirect pathways of allorecognition [9 –11]. We previously demonstrated an increased frequency of allopeptide reactive T cells in the circulation of patients undergoing cellular or chronic, humoral rejection [12– 14]. We postulated that allopeptide reactive T cells differentiate upon recognition of HLA antigens from the injured graft on self APC that have processed donor cells killed via the direct pathway. Such
654
P. Cinti et al. / Human Immunology 70 (2009) 651– 654
Table 2 ACR and AMR in recipients with and without anti-HLA antibodies post-transplantation ACR ⫹ Antibodies to Both HLA class I and II antigens Class I antigens only Class II antigens only No antibodies
Antibodies to HLA to class I antigens No class I antibodies
Antibodies to HLA to class II antigens No class II antibodies
4
AMR ⫹
⫺
0
4
3 0 1 0 8 16 16 16 p ⫽ 0.01
1
⫺
3
4
3 1 24 32
2 1 1 0 0 24 4 28 p ⫽ 0.0002
3 1 24 32
7 0 9 16 16 16 p ⫽ 0.007
7 25 32
3 4 1 24 4 28 p ⫽ 0.03
7 25 32
5 0 11 16 16 16 p ⫽ 0.04
5 27 32
2 3 2 25 4 28 p ⫽ NS
5 27 32
allopeptide reactive T cells produce cytokines that stimulate the maturation of other immunocytes, including alloantibody producing B cells, natural killer cells, macrophages, and dendritic cells. It follows from this scenario that factors facilitating the onset of acute rejection (mediated by T cells with direct allorecognition ability), Table 3 ACR and AMR in patients developing FC-XM and CDC-XM detectable DSA post-transplantation ACR
Post-transplant FC-XM⫹ Negative FC-XM
Post-transplant FCXM⫹ Negative FC-XM
Post-transplant CDC-XM⫹ Negative CDC-XM
Post-transplant CDC-XM⫹ Negative CDC-XM
⫹
⫺
8 8 16 p ⫽ 0.003 AMR
0 13 13
8 21 29
⫹ 4 0 4 p ⫽ 0.003 ACR
⫺ 4 21 25
8 21 29
⫹ 6 9 15 p ⫽ 0.02 AMR
⫺ 0 11 11
6 20 26
⫹ 3 1 4 p ⫽ 0.03
⫺ 3 19 22
6 20 26
such as cross-reactive alloantibodies that opsonize graft cells, may promote inflammation and ACR. It is obvious, however, that early diagnosis and treatment of ACR restores quiescence in most patients, probably by allowing chronic stimulation of noncytolytic CD8⫹ T cells that are MHC class I allorestricted and acquire suppressor function [19]. It is equally important to notice that, in our experience, AMR is a reversible phenomenon when diagnosed and treated in a timely and efficient manner [15–17]. Taken together, our data emphasizes the importance of antibody studies for management of patients receiving organ allografts. Acknowledgements The statistical analysis was supported by the Interuniversitary Organ Transplantation Consortium, Rome, Italy. References [1] Singh N, Pirsch J, Samaniego M. Antibody-mediated rejection: Treatment alternatives and outcomes. Transplant Rev 2009;23:34– 46. [2] Jackson AM, Zachary AA. The problem of transplanting the sensitized patient: Whose problem is it? Front Biosci 2008;13:1396 – 412. [3] Jordan SC, Pescovitz MD. Presensitization: The problem and its management. Clin J Am Soc Nephrol 2006;1:421–32. [4] Zeevi A, Girnita A, Duquesnoy R. HLA antibody analysis: Sensitivity, specificity, and clinical significance in solid organ transplantation. Immunol Res 2006;36: 255– 64. [5] Tinckam KJ, Chandraker A. Mechanisms and role of HLA and non-HLA alloantibodies. Clin J Am Soc Nephrol 2006;1:404 –14. [6] Vasilescu ER, Ho EK, Colovai AI, Vlad G, Foca-Rodi A, Markowitz GS, et al. Alloantibodies and the outcome of cadaver kidney allografts. Hum Immunol 2006;67:597– 604. [7] Ho EK, Vasilescu ER, Colovai AI, Stokes MB, Hallar M, Markowitz GS, et al. Sensitivity, specificity and clinical relevance of different cross-matching assays in deceased-donor renal transplantation. Transpl Immunol 2008;20:61–7. [8] Su¨sal C, Opelz G. Kidney graft failure and presensitization against HLA class I and class II antigens. Transplantation 2002;73:1269 –73. [9] Su¨sal C, Opelz G. Good kidney transplant outcome in recipients with presensitization against HLA class II but not HLA class I. Hum Immunol 2004;65:810 – 6. [10] Liu Z, Colovai AI, Tugulea S, Reed EF, Fisher PE, Mancini D, et al. Indirect recognition of donor HLA-DR peptides in organ allograft rejection. J Clin Invest 1996;98:1150 –7. [11] Ciubotariu R, Liu Z, Colovai AI, Ho E, Itescu S, Ravalli S, et al. Persistent allopeptide reactivity and epitope spreading in chronic rejection of organ allografts. J Clin Invest 1989;101:398 – 405. [12] Renna-Molajoni E, Cinti P, Orlandini AM, Molajoni J, Cocciolo PL, Evangelista B, et al. Contribution of the direct and indirect allorecognition pathway to the rejection of liver allografts. Transplant Proc 1998;30:2138 –9. [13] Renna-Molajoni E, Cinti P, Evangelista B, Orlandini AM, Molajoni J, Cocciolo PL, et al. Role of the indirect recognition pathway in the development of chronic liver allograft rejection. Transplant Proc 1998;30:2140 –1. [14] Renna-Molajoni E, Cinti P, Elia L, Orlandini AM, Cocciolo P, Molajoni J, et al. Mechanism of liver allograft rejection: Indirect allorecognition. Transplant Proc 1999;31:409 –10. [15] Ciubotariu R, Vasilescu R, Ho E, Cinti P, Cancedda C, Poli L, et al. Detection of T suppressor cells in patients with organ allografts. Hum Immunol 2001;62:15– 20. [16] Molajoni ER, Cinti P, Ho E, Evangelista B, Lonardo MT, Rossi M, et al. Allospecific T-suppressor cells in liver transplantation. Transplant Proc 2001;33:1381–3. [17] Renna Molajoni E, Cinti P, Ho E, Evangelista B, Peritore D, Poli L, et al. Allospecific human T suppressor cells in kidney transplantation. Transplant Proc 2001;33:1129 –30. [18] Su¨sal C, D×hler B, Sadeghi M, Ovens J, Opelz G. HLA antibodies and the occurrence of early adverse events in the modern era of transplantation: a collaborative transplant study report. Transplantation 2009;87(9):2367–71. [19] Vlad G, Suciu-Foca N. Resurgence or emergence of CD8⫹ Ts. Hum Immunol 2008;69:679 – 80.