Clinical relevance of pre and post-transplant immune markers in kidney allograft recipients: Anti-HLA and MICA antibodies and serum levels of sCD30 and sMICA

Transplant Immunology 26 (2012) 81–87

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Clinical relevance of pre and post-transplant immune markers in kidney allograft recipients: Anti-HLA and MICA antibodies and serum levels of sCD30 and sMICA Ghasem Solgi a, Daniel Furst b, Joannis Mytilineos b, Gholamreza Pourmand c, Ali Akbar Amirzargar d,⁎ a

Immunology department, Medical school, Hamadan University of Medical Sciences, Hamadan, Iran Department of Transplantation Immunology, Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, University of Ulm & German Red Cross Blood Donor Services BadenWürttemberg-Hessia, Ulm, Germany c Urology Research Center, Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran d Molecular Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 3 September 2011 Received in revised form 27 November 2011 Accepted 5 December 2011 Keywords: HLA and MICA antibodies sCD30 Kidney allograft

a b s t r a c t Background: This retrospective study aims to determine the prognostic values of HLA and MICA antibodies, serum levels of sCD30 and soluble form of MHC class I related chain A (sMICA) in kidney allograft recipients. Methods: Sera samples of 40 living unrelated donor kidney recipients were tested by ELISA and Flow beads techniques for the presence of anti HLA and MICA antibodies and the contents of sCD30 and sMICA. HLA and MICA antibody specification was performed by LABScreen single antigen beads to determine whether the antibodies were directed against donor mismatches. Results: Within first year post operatively 9 of 40 patients (22.5%) showed acute rejection episodes (ARE) that four of them lost their grafts compared to 31 functioning transplants (P = 0.001). The presence of HLA antibodies before and after transplantation was significantly associated with ARE (P = 0.01 and P = 0.02 respectively). Sensitization to HLA class II antigens pre-transplant was strongly associated with higher incidence of ARE (P = 0.004). A significant correlation was found between ARE and appearance of non-donor specific antibodies (P = 0.02). HLA antibody positive patients either before or after transplantation showed lower graft survival rates than those without antibodies during three years follow-up (P = 0.04 and P = 0.02). Anti-MICA antibodies were observed in 8/40(20%) and 5/40(12.5%) of patients pre and post-transplant respectively. Coexistence of HLA and MICA antibodies was shown in 2 of 4 cases with graft loss. A significant increased level of sCD30 at day 14 (P = 0.001) and insignificant decreased levels of sMICA pre and post operatively were detected in rejecting transplants compared to functioning graft group. Conclusion: Our findings support the view that monitoring of HLA and MICA antibodies as well as sCD30 levels early after transplant has predictive value for early and late allograft dysfunctions and the presence of these factors are detrimental to graft function and survival. © 2012 Elsevier B.V. All rights reserved.

1. Introduction There are several immunologic factors associated with the failure of renal allografts including; HLA matching between donor and recipients, sensitization status of the recipient prior transplantation, ability of the recipients to recognize and respond to donor antigens, the presence of T cell activation markers such as sCD30 pre and post transplantation and other unknown factors [1–3]. Precise monitoring of the immune status of transplant recipients using these factors especially antibodies against HLA and non HLA antigens is very crucial to control antibody mediated failures [1,4,5].

⁎ Corresponding author at: Molecular Immunology Research Center, Tehran University of Medical Sciences, Building No.7, Poursina Ave, Tehran, Iran. Tel.: + 98 21 644 32465; fax: + 98 21 66419536. E-mail address: [email protected] (A.A. Amirzargar). 0966-3274/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2011.12.002

Numerous clinical studies have clarified the generation of donor reactive alloantibody after transplantation and significant contribution of these antibodies as well as non donor specific antibodies to the pathobiology of both acute and chronic allograft rejection and to the graft survival [6–8]. Although, HLA antibodies are responsible for a large part of antibody-mediated damage, they do not by themselves explain all antibody mediated graft losses. Among the other possible antibody targets that lead to graft loss, MHC class I related chain A (MICA) as a new target molecules have motivated a growing interest for further investigations and MICA antibodies have been detected to coincide with antibody-mediated rejection of allograft [9–12]. The MICA encoded by a polymorphic gene family located within the HLA class I region of chromosome 6, which determine polymorphic series of antigens similar to HLA [9,13]. These non HLA antigenic determinants are emerging as important polymorphic non-HLA antibody targets in kidney allograft recipients [14]. MICA antigens have

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found to be expressed on endothelial cells, epithelial cells, monocytes, dendritic cells but not on lymphocytes. The presence of MICA antibody in sera of patients who rejected a kidney allograft is of particular interest, because of the contribution of MICA antigens in activating of both cellular and humoral immune responses [4,5,10,15]. Some seminal studies have shown an evident clinical correlation between the presences of preformed anti-MICA antibodies and early immunologic complication in the absence of donor specific HLA antibodies [10,11,16], association of pre sensitization against MICA antigens with an increased frequency of graft loss [12] and a close association between the presence of MICA antibodies and chronic rejection [4] in kidney allograft recipients. Another prognostic markers which allow us to identify of high risk kidney recipients in patients with preformed anti HLA antibodies and/or in patients without anti HLA reactivity is serum content of sCD30 [17,18]. Measurement of sCD30 levels before and after transplantation predicts not only the risk of acute and chronic rejection, but also can contribute to the selection of appropriate immunosuppressive regimen in high risk recipients for the prevention of acute rejection and chronic allograft nephropathy [3,19,20]. 2. Objectives With mounting evidence for prognostic value of HLA antibodies and sCD30, the present retrospective study was designed to determine the predictive power of both pre and post-transplant HLA antibody screening and sCD30 levels along with anti MICA antibodies and serum levels of sMICA antigen in recipients of living unrelated kidney allograft. 3. Patients and methods Pre and post-transplant sera samples from 40 living unrelated donor kidney recipients who consecutively enrolled in our study between June 2006 and July 2007 were assayed retrospectively for immunologic markers including; antibodies against MICA and HLA class I and class II antigens, serum levels of sCD30 and soluble form of MICA (sMICA). This study was approved by research ethics committee from the university. Informed consent was obtained from all patients for the use of data for scientific analysis. Immunosuppressive regimen received by these patients consisted of conventional triple therapy without induction agents (Cyclosporine, Mycophenolate Mophetile and methyl prednisone) which initiated 24 hours before transplantation. Twenty of these patients had received low dose infusion of donor bone marrow cells (2.19 × 109 ± 1.13 × 109 mononuclear cells/recipient) concurrent with renal transplantation in order to augment peripheral microchimerism. The data for microchimerism analysis in this group compared to 20 non-infused patients have been described elsewhere [21]. Herein, we performed a retrospective analysis to determine the prognostic value of the mentioned immunologic markers in those patients who experienced acute rejection episodes (ARE) compared to well functioning graft group during meanly three years post transplant follow up. All patients with ARE were treated with steroid boluses as first line therapy and with ATG in case of steroid resistance. Need for dialysis within first week after surgery was considered as delayed graft function and return to dialysis due to acute rejection, death, transplantectomy was considered as graft loss. HLA genotyping for A, B and DR loci in all patients and donors was done using standard PCR-SSP method (A, B, DR Low resolution SSP-PCR kit, CTS, Heidelberg, Germany). Donor specific WBC cross-match and PRA test was performed by complement-dependent cytotoxicity as routine pre-transplant testing. Negative results for cross-match and PRA percentages less than %10 were considered as no-contraindication to transplantation. However, antibody screening/identification by ELISA or

Luminex methods had not been performed at the time of transplantation. These tests were carried out retrospectively for the purpose of the present study. 3.1. Anti HLA and MICA antibodies screening and identification Sera samples from all patients were screened for the presence of anti HLA class I and class II antibodies before and after transplantation (day 30) by ELISA methods (Ab Screen & Ab Ident, HLA class I and Class II, Biotest AG, Dreieich, Germany) and flow beads techniques (LAB screen mixed and LAB screen single antigen beads for HLA class I and class II antibodies; One Lambda, Canoga Park, CA, USA) according to the manufacturer's protocols. Details for ELISA assay have been described previously by this group [22]. Detection of anti-MICA antibodies was performed by Luminex technology. Pre and post-transplant sera samples were tested by LabScreen Mixed (One Lambda Inc) for screening of anti-MICA antibodies according to the manufacturer's instructions. Positive sera in the screening assay were confirmed by LabScreen MICA single-bead antigen assay (One Lambda Inc) to identify anti-MICA antibody specificity. The single antigen test detects antibodies against a panel of 8 MICA single antigens MICA*001, *002, *004, *007, *009, *012, *017, *018, *019, and *027. The fluorescent signal for each MICA allele–coated bead was measured using LABScan 100 Flow Cytometry (Luminex Corporation) and analyzed by LABScreen v. 3.1 software (One Lambda). The Luminex fluorescence value from the MICA assay for each sera sample was normalized by subtracting the value of normal serum control in the same test set. The cut-off value of 3000 or greater was considered to be positive reaction to the MICA antigen bead. 3.2. MICA allele typing by DNA sequencing method DNA typing of MICA alleles was performed by sequencing of exons 2, 3, 4 and 5 for this locus. Development of sequencing method was previously described [23]. Briefly, MICA locus was initially amplified by two generic PCR using different forward and reverse primers for exon 2–4 and exon 5 separately. PCR products were purified and then reamplified in cycle sequencing manner by specific sequencing primers for each exon. Sequences were read on an ABI-Prism 3730 capillary sequencer (Applied Biosystems) and analysis of the raw sequences was performed using the ‘SEQUENCE PILOT’ software (JSI, Freiburg, Germany). 3.3. Soluble CD30 and soluble MICA measurement Pre and post-transplant sera (days 5, 14 and 30) were assayed for sCD30 levels by ELISA method (Bender Med System, Vienna, Austria). And soluble form of MICA (sMICA) was detected in pre and post transplant (day 30) sera from all patients and by luminex technique using a customized sandwich immunoassay according to the manufacturers protocol (Luminex Corporation). 3.4. Statistical analysis Results are presented as mean ± SD or mean ± SEM. The statistical significance of mean differences was determined by unpaired t-test or Mann–Whitney U test. Contingency table were analyzed for differences in frequencies among categorical variables between patients with acute rejection and functioning graft using fisher's exact test. Regression analysis was done to identify correlations between quantitative markers as prognostic factors for allograft outcomes. Kaplan Meier's estimates and log-rank statistics were used for comparison of patients and graft survival between different groups of patients. A two-tailed P value 0.05 was considered significant. Statistical analysis was done using SPSS version 15 for Windows.

G. Solgi et al. / Transplant Immunology 26 (2012) 81–87 4. Results The demographic data on patients are presented in Table 1. All patients received HLA mismatched kidney transplant from living unrelated donors and only 4 cases were full matched for MICA alleles. There was no statistically significant difference in terms of average number of HLA and MICA mismatches between patients with ARE and those without ARE. Among 40 patients, a total of nine cases (22.5%) showed ARE within four months after transplantation. Four of these rejected cases lost their graft: 2 with irreversible biopsy proved acute rejection, one death with functioning graft occurred at day 35 and one transplantectomy at day 30 was necessary due to the uncontrolled bleeding. Hence, one year graft survival in rejecting group was significantly lower than in patients with well functioning graft (55.5% vs. 100% P = 0.001). Also, one more case with biopsy proved chronic rejection at the end of the second year post transplantation was shown among patients without ARE. Therefore, patients and graft survival rates were 97.5% and 87.5% respectively after meanly three years of follow up. Delayed graft function was observed totally in 7 patients, 4 of them were in the rejecting and 3 in the functioning group respectively (P = 0.03)

4.1. Pre and post-transplant anti-HLA antibodies Merging the results of both ELISA and Luminex methods showed that concordance between both methods for HLA antibody screening is 65% and 54.7% before and after transplant respectively. Hence, the data from both methods for antibody screening was considered for comparison between rejecting and non rejecting patients. Twenty of the 40 patients (50%) were sensitized to HLA antigens prior to transplantation; 15 patients to HLA class I, 14 to HLA class II and 11 cases to both class I and class II antigens. Also, 21 patients (52.5%) showed post-transplant antibodies; 17 to HLA class I, 14 to HLA class II and 12 to both classes of HLA antigens (Table 2). The presence of pre and post transplant antibodies against HLA class I and/or class II was significantly associated with acute rejection episodes (P = 0.01 and P = 0.02, Table 2). Among 11 patients with pre-transplant antibodies against both HLA Class I and Class II antigens, four cases were in the rejecting group that 2 of them lost their graft (2/4 vs. 0/7, P = 0.10). Similarly, among 12 patients with post-transplant antibodies (Class I and II) five cases showed ARE with 3 graft losses (3/5 vs. 0/7, P = 0.04).

Table 1 Demographic and clinical data of all kidney transplant patients (n = 40). Recipient's age in year (Mean ± SD) Recipients gender (M/F) Donor's age in year (Mean ± SD) WBC Cross match Pre-transplant PRA (by CDC) 0–5% 5–10% HLA mismatches (2–6 mM) 2 mM 3 mM 4 mM 5 mM 6 mM MICA mismatches (0–2 mM) 0 mM 1 mM 2 Mm Etiology of ESRD Chronic glomerulonephritis Diabetic nephropathy Polycystic kidney disease Hypertension Renal stone Unknown Cold ischemia time in minutes (Mean ± SD) Warm ischemia time in minutes (Mean ± SD) Delayed Graft Function (DGF)

41.4 ± 10.6 (27 / 13) 33.5 ± 9.2 All Neg 37 (92.5%) 3 (7.5%) 1 (2.5%)a 8 (20.0%)a 11(27.5%) 11 (27.5%) 9 (22.5%) 4 (10.0%)a 11 (27.5%) 25 (62.5%) 2 (5.0%) 11 (27.5%) 4 (10.0%) 10 (25.0%) 4 (10.0%) 9 (22.5%) 51.5 ± 11.6 4.1 ± 0.78 7 (17.5%)

Note: there were no significant differences in terms of all variables between patients with acute rejection episodes (n = 9) and with functioning graft (n = 31). PRA: panel reactive antibody; CDC: complement dependent cytotoxicity; Tx: transplantation; MICA: MHC class I related chain A; ESRD: end stage renal disease; CMV: Cytomegalovirus; DGF: Delayed graft function. a All these cases were in functioning graft patients.

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Table 2 Prevalence of pre and post-transplant HLA antibodies in patients with acute rejection episodes versus functioning graft group.

Pre-Tx HLA Abs HLA class I + IIHLA class II + IClass I + II+ Class I- IIPost-Tx HLA Abs HLA class I + IIHLA class II + IClass I + II+ Class I- IIPre and post-Tx Ab Positives

Rejecting group (n = 9)

Functioning group (n = 31)

P values⁎

8 (88.8%) (5E + L, 3E) 5 (55.5%) 7 (77.7%) 4 (44.4%) 0 (0.0%) 8 (88.8%) (6E + L, 2 L) 7 (77.7%) 6 (66.6%) 5 (55.5%) 0 (0.0%)

12 (38.7%) (8E + L, 4E) 10 (32.3%) 7 (22.6%) 7 (22.6%) 21 (67.7%) 13 (41.9%) (5E + L, 3E, 5 L) 10 (32.3%) 8 (25.8%) 7 (22.6%) 20 (64.5%)

0.019

0.02 0.04 0.09 0.007

7 (77.7%)

6 (19.4%)

0.002

0.25 0.004 0.22 0.0003 0.02

Tx: Transplantation; Abs: antibodies; E: ELISA; L: Luminex. ⁎ Fisher's exact test, 2-tailed P values. The presence of post transplant anti HLA class I or class II antibodies exclusively, was more frequent in patients with ARE than non-rejecting group (7/9 vs. 10/31 for class I, P = 0.02 and 6/9 vs. 8/31 for class II, P = 0.04, Table 2). Also, the presence of pre transplant anti-HLA class II antibodies was strongly associated with higher incidence of ARE (7/9 vs. 7/31, P = 0.004, Table 2). Additionally, the absence of both HLA class I and II antibodies before and after transplantation was more frequent in patients with well functioning graft (P= 0.0003 and P = 0.007 respectively, Table 2). 77.7% of patients with ARE versus 19.4% of patients without rejection showed HLA antibodies before and after transplantation (P = 0.002, Table 2). Overall, 7/40 (17.5%) developed de novo antibodies during the first 3 month after transplantation; one in the rejected and 6 in functioning patients. More importantly, five patients (12.5%) produced donor specific antibodies (DSA) which 3 of them were in rejecting group versus 2 patients in the functioning group (P = 0.06, Table 3). Within this group 2 cases showed antibodies against donor's class I antigens, 2 to class II antigens and one to both class I and II antigens. On the other hand, 10 patients (25%) showed non-DSA after transplantation. Analyzing the relationship between the development of post transplant antibodies and graft outcomes showed a significant association between ARE and production of non-DSA (P = 0.02, Table 3) Among eight antibody positive patients who experienced ARE, four cases lost their grafts. While, no graft loss was observed in 13 antibody positives in the functioning group (P = 0.01). Therefore, one year graft survival among pre and post-transplant antibody positive patients was significantly lower in rejecting group than non-rejecting patients (50% vs. 100% either before or after transplantation, P = 0.001). Totally, after 3 years of follow-up 5 of the 40 patients (12.5%) eventually lost their grafts. All of them were HLA antibody positive pre and post transplant; 4 cases mentioned above and one with biopsy proven chronic rejection. Conversly, among 35 patients who continued to function only seven cases (20%) were HLA antibody positive (P= 0.001). The comparison of meanly three years graft survival between HLA antibody positive and negative patients showed significantly higher survival rate for antibody negative cases before and after transplantation (P = 0.048 and P = 0.022 respectively, Fig. 1).

Table 3 HLA antibody screening and identification by ELISA and Luminex methods in all patients.

By ELISA Pre-Tx Ab positive Post-Tx Ab positive Ab identification DSA Non DSA By Luminex Pre-Tx Ab positive Post-Tx Ab positive Ab identification DSA Non DSA

Rejecting group (n = 9)

Functioning group (n = 31)

P values*

8 (88.8%) 6 (66.6%)

12 (38.7%) 8 (25.8%)

0.01 0.04

2 (22.2%) 6 (66.6%)

2 (6.4%) 3 (9.7%)

0.21 0.001

5 (55.5%) 8 (88.8%)

8 (25.8%) 10 (32.3%)

0.12 0.005

3 (33.3%) 5 (55.5%)

2 (6.4%) 5 (16.1%)

0.06 0.02

Note: the comparison of both method regarding to the presence of pre and post transplant antibody demonstrate higher frequencies of antibody positives, DSA and non-DSA in rejecting group than functioning graft patients.

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Fig. 1. Comparison of the meanly three years Graft survival between HLA Antibody positive and negative patients before and after transplantation.

4.2. Pre and post transplant antibody against MICA antigens Eight of the 40 patients (20%) were sensitized to MICA antigens prior to transplantation that two of them were in the rejecting group (one with graft loss). Also, five patients (12.5%) showed post-transplant antibodies against MICA (one in rejecting group who lost his graft) (Table 4). There was a relationship between the presence of antiMICA antibodies (with or without anti-HLA antibodies) and graft loss in patients with ARE: Two patients with graft loss secondary to ARE had MICA antibodies before or after transplantation (Table 4). Anti-MICA antibody specification among post transplant antibody positives showed that all five cases had non donor specific antibodies (Non-DSA). 3 of 5 patients produced antibody against MICA*001, 004, 012, 018 and 027 antigens. And the remaining two cases showed antibody against MICA*002, 007, 009, 017 and 019 antigens. However, all of these cases were also positive for non DSA against HLA class I or II or

Table 4 Pre and post-transplant anti-MICA antibodies with or without HLA antibodies in patients with ARE compared to non rejecting group.

Pre-Tx MICA Abs MICA + HLA + Post-Tx MICA Abs MICA + HLA + MICA - HLA MICA + HLA MICA - HLA +

Rejecting group (n = 9)

Functioning group (n = 31)

P values⁎

2 (22.2%) 2(22.2%)§ 1 (11.1%) 1 (11.1%)§§ 1 (11.1%) 0 (0.0%) 7 (77.7%)

6 (19.4%) 4 (12.9%) 4 (12.9%) 4 (12.9%) 17 (54.8%) 1 (3.2%) 10 (32.3%)

1.00 0.60 1.00 1.00 0.02 1.00 0.02

Tx, Transplantation; Abs, antibodies. §, one with graft loss; §§, lost his graft. ⁎ Fisher's exact test, 2-tailed P values.

Table 5 MICA antibody specification in patients with post transplant antibody. Identified antibodies

HLA Antibodies DSA or non (Class I &II) DSA

1 (NR)

MICA*008

Class I+

Non DSA

2(NR)

MICA*002

Class I + II+

Non DSA§

3(NR)a

MICA*002 / 021

MICA*002/007/009/ 017/019 MICA*001/004/012/ 018/027 MICA*002/007/009/ 017/019 MICA*001/004/012/ 018/027 MICA*001/004/012/ 018/027

Class II+

Non DSA

Class I + II+

Non DSA§

Class I + II+

DSA

a

MICA*008 / 009

5(AR)

b

MICA*002 / 007

4.3. Serum levels of sCD30 and sMICA Increased levels of sCD30 was shown at days 5, 14 and 30 post operatively in patients with acute rejection episodes compared to functioning transplants and it was only statistically significant at day 14 (P = 0.001, Table 6). Also, we observed higher insignificant levels of sCD30 before transplantation in patients without ARE compared to the rejecting ones (P = 0.09, Table 6). However, all patients who had higher pre-transplant levels of serum sCD30 (> 100 ng/ml), also were positive for HLA antibodies before transplantation. Soluble form of MICA was detected in sera of 31/40 (77.8%) and 30/40 (75%) of patients pre and post-transplant (day 30) respectively. Furthermore, sMICA was not detectable in sera of 2 cases before and 2 other cases after transplantation among patients with ARE. The pre and post-transplant serum levels of sMICA was insignificantly higher in non-rejecting patients than rejecting group (P = 0.33 and P = 0.73 respectively, Table 6). In addition, measuring the serum creatinine concentration as a surrogate marker of early graft dysfunction showed higher levels in patients with ARE during first year post operatively, but significant differences were observed only at days 7, 14 and

Table 6 Pre and post transplant levels of sCD30 and sMICA in patient's serum.

Patients Donor mismatched alleles

4(NR)

both antigens except for one case that had DSA and lost her graft as a consequence of acute rejection (Table 5). As shown in Table 4, the coexistence of both HLA and MICA antibodies was observed in 6/40 (15%) prior to transplantation and more frequent in rejecting patients than functioning group (P = 0.60). Also, a similar frequency for simultaneous presence of both antibodies was found after transplantation between both groups (P = 1.00). 77.7% of the patients with ARE had HLA antibodies in the absence of MICA antibodies compared to 32.3% with functioning graft (P = 0.02, Table 4). The absence of simultaneous HLA and MICA antibodies was observed in a significantly higher frequency among patients without ARE compared to rejecting group after transplantation (P = 0.02, Table 4).

NR, Non-rejected; AR, acute rejection; a, these cases were also positive before transplantation with the same specificities; §, indicate specified antibodies by only luminex method; b, lost its graft secondary to acute rejection and in the presence of donor specific HLA class II antibodies.

Rejecting group (n = 9) sCD30 levels (ng/ml) Pre-Tx (Mean ± SD) Post-Tx , Day 5 (Mean ± SEM) Day 14 (Mean ± SEM) Day 30 (Mean ± SEM) sMICA levels (pg/ml) Pre-Tx (Mean ± SEM) Post-Tx, Day 30 (Mean ± SEM) a

Unpaired t-test. Mann Whitney U test. ⁎ Two-tailed P values. b

Functioning group (n = 31)

P values⁎

57.8 ± 30.3

82.4 ± 56.5

0.09a

63.2 ± 13.7

41.6 ± 6.07

0.15b

46.6 ± 10.6

17.8 ± 7.7

0.001b

39.01 ± 14.8

17.3 ± 4.2

0.22b

24.22 ± 2.58

39.36 ± 6.31

0.33b

15.02 ± 1.48

22.34 ± 2.98

0.73b

G. Solgi et al. / Transplant Immunology 26 (2012) 81–87

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Fig. 2. Correlations between serum creatinine and sCD30 levels (ng/ml) in different time intervals for all patients.

360 compared to the patients without ARE (5.58 ± 3.23 vs. 2.09± 1.19, P = 0.01; 4.55 ± 3.56 vs. 1.86± 0.65, P = 0.05 and 1.62± 0.19 vs. 1.28 ± 0.32, P = 0.01 respectively). Regression analysis revealed the significant positive correlation between serum creatinine levels at day 7 and sCD30 concentrations at days 5 and 14 (P = 0.01 and P = .0.001 respectively), and similarly between sCD30 levels at day 14 and serum creatinine at day 14 and 30 post operatively (P = 0.03 and P = 0.01 respectively) (Fig. 2).

5. Discussion Fine-tuning of the patient's immune function is a major challenge in the treatment and monitoring of allograft recipients, because the capability of predicting immunological complications before detection of clinical parameters can enhance the safety of immunosuppressive therapy protocols [1,4,16]. Antibodies are visible indicators of transplantation antigens and there is accumulating evidence that HLA antibodies can directly cause allograft rejection in humans because of the presence of HLA antibodies before graft failure and in almost all cases after kidney failure [6,16,24]. Moreover, antibodies against non HLA antigens such as MICA have also been recognized to contribute to the pathogenesis of antibody mediated rejection, as both acute and chronic rejections have occurred in the absence of detectable HLA antibodies [16]. There is growing body of data that show a relevant association between the presence of pre and post-transplant MICA antibodies, allograft rejection and lower graft survival in transplant recipients [5,9,11]. In the current study, we found that patients with pre and posttransplant HLA antibodies had a higher incidence of ARE (P = 0.01 and P = 0.02), more graft loss (P = .001), higher levels of sCD30 and

serum creatinine and decreased contents of sMICA early after transplantation as compared to the patients without antibodies. Additionally, antibody positive patients either before or after transplantation showed lower graft survival during meanly 3 years of follow-up (P = 0.04 and P = 0.02 respectively). Patients with ARE showed higher but insignificant frequencies of simultaneous presence of both HLA class I and II antibodies before and after transplantation as compared to the functioning group. Although, more graft loss was observed among these HLA antibody positive patients, it was only significant for the presence of post transplant antibodies in the rejecting group. The presence of post transplant HLA class I or class II antibodies exclusively was more evident in rejecting patients versus functioning group (P = 0.02 and P = 0.04) and this independency for pre transplantation was observed only for the presence of HLA class II antibodies between both groups (P = 0.004). In a previous report from this center (22) on comparison of the early follow-up data between infused patients and controls, lower incidence of ARE (3 vs. 6 cases in controls), low prevalence of DSA (1 vs. 3 cases), decrease in serum creatinine levels and improved allograft function was observed in patients with donor cells infusion. In consistent with the collaborative transplant study data [25,26] we found an association between sensitization against HLA class I and II antigens and higher incidence of rejection of HLA mismatched kidney allograft. Similarly, the pre transplant presence of HLA class II antibodies in the absence of HLA class I did not affect graft survival in kidney recipients. We demonstrated that all of 4 patients with graft loss during first year had HLA antibodies pre and post-transplant compared to 8/31

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(25.8%) of patients with stable graft function (P = 0.009), which is in line with similar studies [2,27,28]. This study depicted that 12.5% of patients had DSA, 27.5% had non DSA and 47.5% had no HLA antibodies post transplant. Among functioning transplants, 6.4% showed DSA which is in agreement with the findings of Cardarelli et al. [28]. Meanwhile, acute rejection was associated significantly with the presence of non-DSA (P = 0.02, Table 3). It is note-worthy that despite considering the results of both ELISA and flow bead methods, a lower frequency of both DSA and non DSA was found in our patients. In comparison, Zhang and Piazza studies [24,29,30] reported a higher prevalence of DSA (22.4% and 24.2% respectively) and non DSA (38.8%) in kidney allograft patients within the first year after transplantation. 17.5% of the patients developed de novo antibodies during the first 3 months after transplantation; one in rejected and 6 in functioning graft patients. Similarly, a cooperative study by Terasaki et al. [8] showed de novo production of HLA antibodies in 14.7% of patients within first year post operatively. As expected [30,31], the presence of HLA antibodies correlated with higher incidence of ARE, more allograft failure, increased serum creatinine levels and poorer outcomes compared to those patients without antibodies. In accordance with two other studies [16,32] on reporting higher frequencies of failure among antibody positive patients before or after transplantation, we found that all five patients who lost their grafts had HLA antibodies either before or after transplantation. With reference to anti-MICA antibodies, 20% and 12.5% of patients were positive pre and post transplant respectively. Also, more prevalence of coexistent MICA and HLA antibodies before transplantation was found in rejecting group versus functioning group but not statistically significant, maybe due to the small number of patients in this study. These results are supported by similar studies [5,9,16] on demonstrating the higher frequency of ARE and the graft loss in the presence of both MICA and HLA antibodies. More importantly, occurrence of graft loss secondary to acute rejection was accompanied with the presence of antibodies against MICA either before or after transplantation. The appearance of MICA antibodies in rejecting patients was observed only in two cases pre transplant (one with graft loss) and in one patient after transplant (who lost its graft) and interestingly all 3 cases were positive for HLA antibodies as well. In other words, simultaneous presence of HLA and MICA antibodies could be more predictive for graft loss in patients who experienced acute rejection episodes. In spite of donor mismatches for MICA antigens, none of antibody positive patients showed DSA against these antigens (Table 5). It has been shown that donor specific antibodies against either HLA or MICA antigens are often observed in situ in the organ and when saturated, spill out into the peripheral circulation [5,16,33]. Accordingly, we found higher prevalence of non DSA against HLA and MICA antigens in our patients particularly in those with acute rejection episodes. Hence, regardless of HLA or MICA antibody specificity and whether or not the antibodies are against donor derived antigens, it is reasonable to predict that their appearance after transplant is detrimental to the allograft [34]. Another risk factor that could be remarkably predictive for allograft outcome is pre and post transplant levels of sCD30 molecule. Several studies have reported that elevated sCD30 levels before and after transplant are associated with poorer allograft outcomes [19,20,35,36]. Our findings are supported by these evidences and as expected [37,38], we did observe that patients with ARE had higher post transplant sCD30 levels (day 14) than patients without ARE (P = 0.001). Moreover, sCD30 content was also higher but insignificantly at days 5 and 30 compared to non rejecting patients. Compared to pre-transplant levels, the post transplant sCD30 contents generally decreased except for rejecting patients who had higher levels at day 5 than non rejecting patients. This is in agreement with the results of study by Pelzl et al. [17,36].

It is speculative that, in the presence of HLA-DR mismatches and consequent stimulation of recipient T cells, the expression of membrane CD30 is up regulated on antigen specific CD4+ T cells and thus the blockade of CD30–CD30L by sCD30 occurs which is favoring the development of aggressive Th1 response rather than Th2 response [39,40]. Surprisingly, patients with functioning graft showed a slightly higher concentration of sCD30 before transplantation but insignificantly as compared to rejecting patients. Regarding to the sMICA levels, almost a similar frequency for the presence of sMICA was found before and after transplant for all patients (77.8% and 75% of patients respectively). Consistent with the finding of Suarez et al. study [41], patients who experienced ARE showed lower levels of sMICA either before or after transplant compared with functioning group. A possible explanation for the contribution of sMICA in better allograft outcomes could be down regulation of NKG2D surface expression and consequent functional impairment of CD8+ T cells and NK mediated cytolysis [9]. Finally, the regression analysis showed that increasing levels of serum creatinine could be predictive for increase in sCD30 levels during the first month after transplantation which both of them are prognostic for poorer outcomes in kidney allograft recipients. Notably, almost all cases with higher concentrations of creatinine and sCD30 showed poorer outcome than the other group so that, four patients with graft loss and also with ARE were in this group. Furthermore, among the remaining five cases with ARE, four patients showed a remarkable increase in both prognostic markers compared to the other group. It is of note that two patients with low serum creatinine but high concentration of sCD30 also experienced reversible acute rejection episodes. Altogether, our findings indicate that the presence of both HLA and MICA antibodies before and after transplant are detrimental for allograft function and survival. In the same way, the presence of HLA class II antibodies pre transplantation is more predictive for acute rejection and poor prognosis. Patients who showed coexistence of both HLA and MICA antibodies lost their graft secondary to acute rejection. These findings are in line with the humoral theory of transplantation that proposed by Terasaki PI [6,42]. In conclusion, this study support the view that monitoring of kidney transplant recipients for antibodies against HLA and MICA antigens besides measuring the sCD30 and sMICA levels early after transplantation are noninvasive approaches to assist in diagnosis of acute rejection episodes as well as the identification of patients at risk of early graft dysfunction. Conflict of interest The authors have no conflicting interest for this study. Acknowledgments The authors gratefully thank all staff members of the transplantation ward and the urology research center from Sina Hospital for their excellent assistance in the pre and post-transplantation sampling and for providing clinical information during follow up. Also, we thank Luzia for her assistance in performing the flow bead testing. References [1] Süsal C, Pelzl S, Simon T, Opelz G. Advances in pre and posttransplant immunologic testing in kidney transplantation. Transplant Proc 2004;36:29–34. [2] Worthington JE, Martin S, Barker AJ, McWilliam LJ, Dyer PA. The role of HLAspecific antibodies in kidney transplant rejection: published studies and local data. Clin Transpl 2006;20:349–61. [3] Rodríguez LM, París SC, Arbeláez M, Cotes JM, Süsal C, Torres Y, et al. Kidney graft recipients with pretransplantation HLA CLASS I antibodies and high soluble CD30 are at high risk for graft loss. Hum Immunol 2007;68:652–60. [4] Terasaki PI, Ozawa M, Castro R. Four-year follow-up of a prospective trial of HLA and MICA antibodies on kidney graft survival. Am J Transplant 2007;7:408–15.

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