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Blood and intragraft CD27 gene expression in cardiac transplant recipients
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Clinical Immunology 107 (2003) 60 – 64
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Blood and intragraft CD27 gene expression in cardiac transplant recipients Andrey Morgun,a,*,1 Natalia Shulzhenko,a,1 Gisele F. Rampim,a Angela P. Chinellato,a Rosiane V.Z. Diniz,b Dirceu R. Almeida,b Marcia M. Souza,c Marcello Franco,c and Maria Gerbase-DeLimaa a
Division of Immunogenetics, Department of Pediatrics, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil b Division of Cardiology, Department of Medicine, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil c Department of Pathology, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil Received 17 June 2002; accepted with revision 12 December 2002
Abstract The present study investigated gene expression of costimulatory molecule CD27 in relation to the occurrence of acute cardiac rejection. CD27 transcripts were measured by means of quantitative competitive reverse transcriptase–polymerase chain reaction in 120 endomyocardial biopsies and in 89 samples of blood mononuclear cells from 31 recipients. Higher levels of CD27 transcripts were observed in biopsies with rejection than in samples without rejection (medians, 7.1 and 1.9; P ⫽ 0.06). In contrast, blood mononuclear cells collected during rejection showed lower levels than blood mononuclear cells from rejection-free periods (medians, 3.3 vs. 7.9; P ⫽ 0.03). Considering only endomyocardial biopsies without rejection, the values were lower in samples from recipients who did not present any rejection during the first 6 months after transplantation than in those from recipients who had at least one rejection during the same period (medians, 0 vs. 3.5, P ⬍ 0.001; percentage of biopsies expressing CD27, 44% vs. 77%). In conclusion, the presence of intragraft CD27 mRNA may identify recipients at risk for developing acute rejection. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Cardiac allograft rejection; Immunologic monitoring; CD27; Costimulatory molecules; mRNA
Introduction CD27 is a glycoprotein of the tumor necrosis factor receptor family expressed on the surface of 75% of T and 25% of B lymphocytes in peripheral blood [1,2]. Its expression is markedly upregulated during naive T-cell activation and is lost after lymphocyte clonal expansion. Crosslinking of CD27 by its ligand (CD70) provides a costimulatory signal for proliferation and cytokine production by naive T cells and, to a much lesser extent, by memory cells [3]. CD27/CD70 interaction was proposed to be involved in crosstalk between naive and primed lymphocytes, since * Corresponding author. Rua Napolea˜o de Barros 1038, Sa˜o Paulo, SP 04024-003, Brazil. Fax: ⫹55-11-5081-5028. E-mail address: [email protected] (A. Morgun). 1 These authors equally contributed to the work.
CD70 is found on the surface of T and B cells later after activation and in memory cells [4]. Costimulation is a critical event for alloimmunity. It enables a progression of the immune response and prevents lymphocyte apoptosis [5]. Therefore, costimulatory molecules are promising targets of immunotherapy. The costimulatory molecule CD27 has been studied in in vitro and in vivo allogeneic experimental models. Brown et al. [6] showed that CD27/CD70 interaction can selectively enhance the in vitro differentiation of alloantigen-specific cytotoxic T cells. In vivo, CD27/CD70 blockade by anti-CD70 monoclonal antibody prolonged cardiac allograft survival in wild-type mice, and in CD28 knockout recipients, the same treatment resulted in indefinite organ acceptance [7]. In contrast, in islet transplantation anti-CD70 monoclonal antibody administration did not improve allograft survival [8]. In clinical transplantation, the presence of CD27-positive
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A. Morgun et al. / Clinical Immunology 107 (2003) 60 – 64
lymphocytes in blood of liver allograft recipients has been shown to correlate with acute rejection [9]. In addition, two studies have demonstrated elevated serum levels of soluble CD27 during renal and cardiac acute rejection [10,11]. Preliminary data from our laboratory suggested a relationship between intragraft CD27 gene expression and acute cardiac rejection [12]. The purpose of the present study was to further investigate this phenomenon in a larger sample of endomyocardial biopsies (EMB), as well as to evaluate CD27 mRNA expression in the blood of cardiac transplant recipients.
Materials and methods Patients The study comprised 31 cardiac allograft recipients transplanted at Sa˜ o Paulo Hospital, Federal University of Sa˜ o Paulo. The protocol was approved by the Ethics Committee of the Federal University of Sa˜ o Paulo and informed consent was obtained from all subjects under study. All recipients were adults maintained on standard triple therapy immunosuppression. Treatment for rejection consisted of pulse therapy with methylprednisolone (1 g daily for 3 days) and/or augmentation of the oral doses of prednisone and cyclosporine. EMB for rejection monitoring were routinely performed during the first 6 months after transplantation according to a standard schedule (weekly during the first month, every 15 days during the second month, and monthly thereafter). Rejection was graded according to the scoring scheme of the International Society for Heart and Lung Transplantation after the examination of three or four fragments of each biopsy. Biopsies One fragment of the biopsy specimen was snap-frozen and stored in liquid nitrogen until the time for mRNA extraction. A total of 120 biopsies were included in the study. Blood mononuclear cells (BMC) Mononuclear cells were separated (Ficoll-Hypaque) from 5 ml of heparinized blood collected at the time of cardiac catheterization during regular biopsies. BMC were kept frozen in 10% dimethyl sulfoxide solution until the time of mRNA extraction. A total of 89 BMC samples were included in the study. Reverse transcription–polymerase chain reaction Gene expression was evaluated by quantitative-competitive reverse transcriptase–polymerase chain reaction (RTPCR). RNA isolation, reverse transcription, PCR mixture,
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primer design, and competitor construction were performed as previously described in detail [13]. The primers for CD27 were S: 5⬘-AGGGACAAGGAGTGCACCGAGT-3⬘; and AS: 5⬘-TGCTTCCCACTCTCCACCTCATC-3⬘ (product size, 646 bp). Denaturation, annealing, and extension conditions were 94°C for 45 s, 59°C for 30 s, and 72°C for 30 s, respectively. Forty cycles of PCR were carried out in an MJ Research PTC-200 Thermocycler (Watertown, MA). The primers and conditions for glyceraldehyde-3-phosphate dehydrogenase mRNA were as previously described [13]. The PCR products were separated by electrophoresis on ethidium bromide-stained 2% agarose gels and the band intensities were analyzed by using the Kodak Digital Science-EDAS 120 system (Eastman-Kodak Co., Rochester, NY). Each sample was tested in duplicate. The cDNAs derived from the samples were coamplified with a known amount of the competitor DNA. The transcript levels were quantified as previously described [13]. Statistical analysis Quantitative data were analyzed statistically by nonparametric Mann-Whitney test for unpaired samples, by Wilcoxon test for paired samples, and Spearman correlation test. Fisher exact test was used for qualitative data analysis. Positive and negative predictive values for the development of rejection were calculated by using receiver operating characteristic curve analysis (MedCalc software, Mariakerke, Belgium), considering a prevalence of 50%.
Results According to the presence of at least one biopsy graded ⱖ1B during the first 6 months after transplantation, the recipients were classified as rejectors (n ⫽ 24) and nonrejectors (n ⫽ 7). In the group of rejectors, higher levels of CD27 mRNA were detected in EMB with rejection ⱖ1B (n ⫽ 36) than in the chronologically closest EMB graded 0 –1A (medians, 7.1 vs. 1.9, respectively; P ⫽ 0.06) (Fig. 1a). In contrast, BMC collected during rejection (n ⫽ 26) presented lower levels of CD27 transcripts than the chronologically closest samples collected during a quiescent period (medians, 3.3 vs. 7.9, respectively; P ⫽ 0.03) (Fig. 1b). Sixty-three percent of grade 0 –1A EMB were collected before rejection (median of 28 days before), and the remaining were collected after rejection (median of 27 days after). CD27 mRNA levels did not differ significantly between these two groups. CD27 mRNA levels in EMB without rejection correlated negatively with the number of days between no rejection and rejection (r ⫽ ⫺0.32, P ⬍ 0.02; Fig. 2). Concerning BMC, 69% of samples without rejection were collected, in median, 43 days before rejection and the
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Fig. 1. Individual values of CD27 mRNA expression during periods with (R) and without (no R) acute cardiac rejection in recipients who presented acute rejection in the first 6 months after transplantation (a) in endomyocardial biopsies and (b) in blood mononuclear cells. Horizontal lines represent the median values for each group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
remaining BMC were collected, in median, 21 days after rejection. CD27 mRNA levels did not differ significantly between samples preceding or following rejection. In contrast to EMB, in BMC samples there was no correlation between CD27 mRNA expression levels during the no rejection period and the number of days apart from rejection. Comparing samples without rejection from nonrejectors and rejectors, lower values in EMB were detected in the former group, with medians of 0 vs. 3.5 (P ⬍ 0.001) (Fig. 3a), whereas the difference between the corresponding levels in BMC was not statistically significant (Fig. 3b). Furthermore, CD27 transcripts were detected in 44% of EMB without rejection from nonrejectors and in 77% of the same grade EMB from rejectors (P ⬍ 0.02). Positive and negative predictive values of CD27 mRNA detection in grade 0 –1A biopsies for acute rejection development were 64% and 71%, respectively.
Discussion Acute rejection is still an important obstacle in clinical cardiac transplantation. It is becoming evident that posttransplant immunological monitoring of cardiac allograft recipients has a great potential to contribute to the diagnosis of rejection, for the assessment of the individual risk of rejection, as well as to provide new insights into the immune response against the graft, which might lead to new approaches to prophylactic or therapeutic interventions [14]. The present study showed that during rejection there was an increase in intragraft and a decrease in blood CD27 mRNA levels. In addition, higher intragraft CD27 levels were observed during rejection-free periods in recipients that presented at least one rejection episode during the first 6 months after transplantation than in those who did not present any rejection during this period.
Fig. 2. Correlation between CD27 mRNA levels in endomyocardial biopsies without rejection (grade 0 –1A) and number of days apart from rejection. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 3. Individual values of CD27 mRNA expression (a) in endomyocardial biopsies and (b) in blood mononuclear cells collected during the periods without rejection from recipients who experienced (Rejectors) or did not experience (Non-rejectors) acute rejection in the first 6 months after transplantation. Horizontal lines represent the median values in each group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
The intragraft increase of CD27 mRNA levels during rejection parallels the findings regarding other immune activation molecules such as costimulatory molecules (TIRC7 and CD40L), cytokines (IL-2, IL-4, IFN-␥, and IL-8), cell adhesion molecules (VCAM1 and ICAM1), and cytotoxic T-cell molecules (granzyme, perforin, and FasL) [13,15– 20]. The increase in CD27 expression was not detected 7–15 days before appearance of histological alterations (data not shown), as was previously shown for TIRC7, another costimulatory molecule [13]. However, a weak but significant negative correlation between intragraft CD27 mRNA levels in the samples without rejection and the time interval between no rejection and rejection was detected. One possible explanation for these observations could be a relatively high constitutive expression of CD27 on naive T cells [1]. In contrast to CD27, its ligand, CD70, does not appear to be expressed in EMB during periods with or without rejection [12]. This finding, along with the fact that CD70 is preferentially expressed on chronically activated T cells, leads to the speculation that acute rejection is driven rather by an activation of naive T cells than by a reactivation of primed lymphocytes. In contrast to the increased intragraft CD27 mRNA expression, its level in BMC was lower during rejection than during quiescent periods. This finding is in agreement with our studies regarding blood kinetics of mRNA expression of other Tcell activation molecules [21] and our hypothesis to explain this phenomenon is the homing of activated T cells through the blood to the site of rejection [13]. Since soluble CD27 is released by activated T cells, the decline in serum levels of soluble CD27 during acute renal and cardiac rejection, as described in two separate studies [10,11], is in accordance with our findings of decreased CD27 mRNA levels in the blood during rejection. In an allograft recipient, there is a continuous balance between alloimmunity and immunosuppression, the former leading to rejection and the latter allowing the acceptance of the graft. Nevertheless, an alloimmune response seems to
continue even during apparently rejection-free periods. In the present study, we demonstrated that this “subclinical” immune response may be detected by intragraft CD27 mRNA measurements. Indeed, biopsies without any evidence of histological rejection from patients that experienced a rejection (rejectors) showed increased CD27 expression compared to the samples from nonrejectors. No reports on similar markers of risk of rejection have been published, with the exception of the work by De GrootKruseman et al. [22] where high levels of basic fibroblast growth factor mRNA in the first week EMB were associated with acute rejection during the first year following cardiac transplantation. These findings suggest that rejectors present higher levels of alloimmune response even during histologically quiescent periods. Although intragraft CD27 levels alone could not be considered as a marker of rejection risk in the clinical setting, its analysis in combination with other markers, for example basic fibroblast growth factor mRNA, could strengthen the predictive value of molecular diagnosis. In conclusion, the present study has shown that intragraft CD27 gene expression level increases during ongoing rejection and that the serial evaluation of its level during rejection-free periods could be helpful for the discrimination of recipients at high and low risk for presenting acute rejection during the first 6 months after cardiac transplantation. These data are relevant not only to the understanding of some of the mechanisms underlying cardiac allograft acute rejection, but also may contribute to the clinical management of cardiac allograft recipients.
Acknowledgments Supported by Fundac¸ a˜ o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo-FAPESP, and Conselho Nacional de Desenvolvimento Cientifico e Tecnolo´ gico-CNPq. We would like
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to thank Adalberto Socorro da Silva and Elizabeth Cristina P. Hurtado for technical assistance.
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[12] N. Shulzhenko, A. Morgun, A.P. Chinellato, G.F. Rampim, R.V.Z. Diniz, D.R. Almeida, M. Gerbase-DeLima, CD27 but not CD70 and 4-1BB intragraft gene expression is a risk factor for acute cardiac allograft rejection in humans, Transplant. Proc. 34 (2002) 474 – 475. [13] N. Shulzhenko, A. Morgun, G.F. Rampim, M. Franco, D.R. Almeida, R.V.Z. Diniz, A.C.C. Carvalho, M. Gerbase-DeLima, Monitoring of intragraft and peripheral blood TIRC7 expression as a diagnostic tool for acute cardiac rejection in humans, Hum. Immunol. 62 (2001) 342–347. [14] C.C. Baan, W. Weimar, Intragraft cytokine gene expression: implications for clinical transplantation, Transplant. Int. 11 (1998) 169 – 180. [15] N. Shulzhenko, A. Morgun, M. Franco, M.M. Souza, D.R. Almeida, R.V.Z. Diniz, A.C.C. Carvalho, A. Pacheco-Silva, M. Gerbase-DeLima, Expression of CD40 ligand, interferon-␥ and Fas ligand genes in endomyocardial biopsies of human cardiac allografts: correlation with acute rejection, Braz. J. Med. Biol. Res. 34 (2001) 779 –784. [16] C.C. Baan, N.E.M. van Emmeric, A.H.M.M. Balk, W.G.V. Quint, B. Mochtar, N.H.P.M. Jutte, H.G.M. Niester, W. Weimar, Cytokine mRNA expression in endomyocardial biopsies during acute rejection from human heart transplants, Clin. Exp. Immunol. 97 (1994) 293– 298. [17] X.P. Zhang, S.E. Kelemen, H.J. Eisen, Quantitative assessment of cell adhesion molecule gene expression in endomyocardial biopsy specimens from cardiac transplant recipients using competitive polymerase chain reaction, Transplantation 70 (2000) 505–513. [18] N. Shulzhenko, A. Morgun, X.X. Zheng, R.V.Z. Diniz, D.R. Almeida, N. Ma, T.B. Strom, M. Gerbase-DeLima, Intragraft activation of genes encoding cytotoxic T lymphocyte effector molecules precedes the histological evidence of rejection in human cardiac transplantation, Transplantation 72 (2001) 1705–1708. [19] S. Alpert, N.P. Lewis, M. Fowler, H.A. Valantine, The relationship of granzyme A and perforin expression to cardiac allograft rejection and dysfunction, Transplantation 60 (1995) 1478 –1485. [20] S.I. Oh, I.W. Kim, H.C. Jung, J.W. Seo, I.H. Chae, H.S. Kim, B.H. Oh, Correlation of Fas and Fas ligand expression with rejection status of transplanted heart in human, Transplantation 71 (2001) 906 –909. [21] A. Morgun, N. Shulzhenko, R.V.Z. Diniz, D.R. Almeida, A.C.C. Carvalho, M. Gerbase-DeLima, Cytokine and TIRC7 mRNA expression during acute rejection in cardiac allograft recipients, Transplant. Proc. 33 (2001) 1610 –1611. [22] H.A. De Groot-Kruseman, C.C. Baan, E.H. Loonen, W.M. Mol, H.G. Niesters, A.P. Maat, A.H. Balk, W. Weimar, Failure to down-regulate intragraft cytokine mRNA expression shortly after clinical heart transplantation is associated with high incidence of acute rejection, J. Heart Lung Transplant. 20 (2001) 503–510.
Clinical Immunology 107 (2003) 60 – 64
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Blood and intragraft CD27 gene expression in cardiac transplant recipients Andrey Morgun,a,*,1 Natalia Shulzhenko,a,1 Gisele F. Rampim,a Angela P. Chinellato,a Rosiane V.Z. Diniz,b Dirceu R. Almeida,b Marcia M. Souza,c Marcello Franco,c and Maria Gerbase-DeLimaa a
Division of Immunogenetics, Department of Pediatrics, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil b Division of Cardiology, Department of Medicine, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil c Department of Pathology, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil Received 17 June 2002; accepted with revision 12 December 2002
Abstract The present study investigated gene expression of costimulatory molecule CD27 in relation to the occurrence of acute cardiac rejection. CD27 transcripts were measured by means of quantitative competitive reverse transcriptase–polymerase chain reaction in 120 endomyocardial biopsies and in 89 samples of blood mononuclear cells from 31 recipients. Higher levels of CD27 transcripts were observed in biopsies with rejection than in samples without rejection (medians, 7.1 and 1.9; P ⫽ 0.06). In contrast, blood mononuclear cells collected during rejection showed lower levels than blood mononuclear cells from rejection-free periods (medians, 3.3 vs. 7.9; P ⫽ 0.03). Considering only endomyocardial biopsies without rejection, the values were lower in samples from recipients who did not present any rejection during the first 6 months after transplantation than in those from recipients who had at least one rejection during the same period (medians, 0 vs. 3.5, P ⬍ 0.001; percentage of biopsies expressing CD27, 44% vs. 77%). In conclusion, the presence of intragraft CD27 mRNA may identify recipients at risk for developing acute rejection. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Cardiac allograft rejection; Immunologic monitoring; CD27; Costimulatory molecules; mRNA
Introduction CD27 is a glycoprotein of the tumor necrosis factor receptor family expressed on the surface of 75% of T and 25% of B lymphocytes in peripheral blood [1,2]. Its expression is markedly upregulated during naive T-cell activation and is lost after lymphocyte clonal expansion. Crosslinking of CD27 by its ligand (CD70) provides a costimulatory signal for proliferation and cytokine production by naive T cells and, to a much lesser extent, by memory cells [3]. CD27/CD70 interaction was proposed to be involved in crosstalk between naive and primed lymphocytes, since * Corresponding author. Rua Napolea˜o de Barros 1038, Sa˜o Paulo, SP 04024-003, Brazil. Fax: ⫹55-11-5081-5028. E-mail address: [email protected] (A. Morgun). 1 These authors equally contributed to the work.
CD70 is found on the surface of T and B cells later after activation and in memory cells [4]. Costimulation is a critical event for alloimmunity. It enables a progression of the immune response and prevents lymphocyte apoptosis [5]. Therefore, costimulatory molecules are promising targets of immunotherapy. The costimulatory molecule CD27 has been studied in in vitro and in vivo allogeneic experimental models. Brown et al. [6] showed that CD27/CD70 interaction can selectively enhance the in vitro differentiation of alloantigen-specific cytotoxic T cells. In vivo, CD27/CD70 blockade by anti-CD70 monoclonal antibody prolonged cardiac allograft survival in wild-type mice, and in CD28 knockout recipients, the same treatment resulted in indefinite organ acceptance [7]. In contrast, in islet transplantation anti-CD70 monoclonal antibody administration did not improve allograft survival [8]. In clinical transplantation, the presence of CD27-positive
1521-6616/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1521-6616(02)00047-5
A. Morgun et al. / Clinical Immunology 107 (2003) 60 – 64
lymphocytes in blood of liver allograft recipients has been shown to correlate with acute rejection [9]. In addition, two studies have demonstrated elevated serum levels of soluble CD27 during renal and cardiac acute rejection [10,11]. Preliminary data from our laboratory suggested a relationship between intragraft CD27 gene expression and acute cardiac rejection [12]. The purpose of the present study was to further investigate this phenomenon in a larger sample of endomyocardial biopsies (EMB), as well as to evaluate CD27 mRNA expression in the blood of cardiac transplant recipients.
Materials and methods Patients The study comprised 31 cardiac allograft recipients transplanted at Sa˜ o Paulo Hospital, Federal University of Sa˜ o Paulo. The protocol was approved by the Ethics Committee of the Federal University of Sa˜ o Paulo and informed consent was obtained from all subjects under study. All recipients were adults maintained on standard triple therapy immunosuppression. Treatment for rejection consisted of pulse therapy with methylprednisolone (1 g daily for 3 days) and/or augmentation of the oral doses of prednisone and cyclosporine. EMB for rejection monitoring were routinely performed during the first 6 months after transplantation according to a standard schedule (weekly during the first month, every 15 days during the second month, and monthly thereafter). Rejection was graded according to the scoring scheme of the International Society for Heart and Lung Transplantation after the examination of three or four fragments of each biopsy. Biopsies One fragment of the biopsy specimen was snap-frozen and stored in liquid nitrogen until the time for mRNA extraction. A total of 120 biopsies were included in the study. Blood mononuclear cells (BMC) Mononuclear cells were separated (Ficoll-Hypaque) from 5 ml of heparinized blood collected at the time of cardiac catheterization during regular biopsies. BMC were kept frozen in 10% dimethyl sulfoxide solution until the time of mRNA extraction. A total of 89 BMC samples were included in the study. Reverse transcription–polymerase chain reaction Gene expression was evaluated by quantitative-competitive reverse transcriptase–polymerase chain reaction (RTPCR). RNA isolation, reverse transcription, PCR mixture,
61
primer design, and competitor construction were performed as previously described in detail [13]. The primers for CD27 were S: 5⬘-AGGGACAAGGAGTGCACCGAGT-3⬘; and AS: 5⬘-TGCTTCCCACTCTCCACCTCATC-3⬘ (product size, 646 bp). Denaturation, annealing, and extension conditions were 94°C for 45 s, 59°C for 30 s, and 72°C for 30 s, respectively. Forty cycles of PCR were carried out in an MJ Research PTC-200 Thermocycler (Watertown, MA). The primers and conditions for glyceraldehyde-3-phosphate dehydrogenase mRNA were as previously described [13]. The PCR products were separated by electrophoresis on ethidium bromide-stained 2% agarose gels and the band intensities were analyzed by using the Kodak Digital Science-EDAS 120 system (Eastman-Kodak Co., Rochester, NY). Each sample was tested in duplicate. The cDNAs derived from the samples were coamplified with a known amount of the competitor DNA. The transcript levels were quantified as previously described [13]. Statistical analysis Quantitative data were analyzed statistically by nonparametric Mann-Whitney test for unpaired samples, by Wilcoxon test for paired samples, and Spearman correlation test. Fisher exact test was used for qualitative data analysis. Positive and negative predictive values for the development of rejection were calculated by using receiver operating characteristic curve analysis (MedCalc software, Mariakerke, Belgium), considering a prevalence of 50%.
Results According to the presence of at least one biopsy graded ⱖ1B during the first 6 months after transplantation, the recipients were classified as rejectors (n ⫽ 24) and nonrejectors (n ⫽ 7). In the group of rejectors, higher levels of CD27 mRNA were detected in EMB with rejection ⱖ1B (n ⫽ 36) than in the chronologically closest EMB graded 0 –1A (medians, 7.1 vs. 1.9, respectively; P ⫽ 0.06) (Fig. 1a). In contrast, BMC collected during rejection (n ⫽ 26) presented lower levels of CD27 transcripts than the chronologically closest samples collected during a quiescent period (medians, 3.3 vs. 7.9, respectively; P ⫽ 0.03) (Fig. 1b). Sixty-three percent of grade 0 –1A EMB were collected before rejection (median of 28 days before), and the remaining were collected after rejection (median of 27 days after). CD27 mRNA levels did not differ significantly between these two groups. CD27 mRNA levels in EMB without rejection correlated negatively with the number of days between no rejection and rejection (r ⫽ ⫺0.32, P ⬍ 0.02; Fig. 2). Concerning BMC, 69% of samples without rejection were collected, in median, 43 days before rejection and the
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Fig. 1. Individual values of CD27 mRNA expression during periods with (R) and without (no R) acute cardiac rejection in recipients who presented acute rejection in the first 6 months after transplantation (a) in endomyocardial biopsies and (b) in blood mononuclear cells. Horizontal lines represent the median values for each group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
remaining BMC were collected, in median, 21 days after rejection. CD27 mRNA levels did not differ significantly between samples preceding or following rejection. In contrast to EMB, in BMC samples there was no correlation between CD27 mRNA expression levels during the no rejection period and the number of days apart from rejection. Comparing samples without rejection from nonrejectors and rejectors, lower values in EMB were detected in the former group, with medians of 0 vs. 3.5 (P ⬍ 0.001) (Fig. 3a), whereas the difference between the corresponding levels in BMC was not statistically significant (Fig. 3b). Furthermore, CD27 transcripts were detected in 44% of EMB without rejection from nonrejectors and in 77% of the same grade EMB from rejectors (P ⬍ 0.02). Positive and negative predictive values of CD27 mRNA detection in grade 0 –1A biopsies for acute rejection development were 64% and 71%, respectively.
Discussion Acute rejection is still an important obstacle in clinical cardiac transplantation. It is becoming evident that posttransplant immunological monitoring of cardiac allograft recipients has a great potential to contribute to the diagnosis of rejection, for the assessment of the individual risk of rejection, as well as to provide new insights into the immune response against the graft, which might lead to new approaches to prophylactic or therapeutic interventions [14]. The present study showed that during rejection there was an increase in intragraft and a decrease in blood CD27 mRNA levels. In addition, higher intragraft CD27 levels were observed during rejection-free periods in recipients that presented at least one rejection episode during the first 6 months after transplantation than in those who did not present any rejection during this period.
Fig. 2. Correlation between CD27 mRNA levels in endomyocardial biopsies without rejection (grade 0 –1A) and number of days apart from rejection. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 3. Individual values of CD27 mRNA expression (a) in endomyocardial biopsies and (b) in blood mononuclear cells collected during the periods without rejection from recipients who experienced (Rejectors) or did not experience (Non-rejectors) acute rejection in the first 6 months after transplantation. Horizontal lines represent the median values in each group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
The intragraft increase of CD27 mRNA levels during rejection parallels the findings regarding other immune activation molecules such as costimulatory molecules (TIRC7 and CD40L), cytokines (IL-2, IL-4, IFN-␥, and IL-8), cell adhesion molecules (VCAM1 and ICAM1), and cytotoxic T-cell molecules (granzyme, perforin, and FasL) [13,15– 20]. The increase in CD27 expression was not detected 7–15 days before appearance of histological alterations (data not shown), as was previously shown for TIRC7, another costimulatory molecule [13]. However, a weak but significant negative correlation between intragraft CD27 mRNA levels in the samples without rejection and the time interval between no rejection and rejection was detected. One possible explanation for these observations could be a relatively high constitutive expression of CD27 on naive T cells [1]. In contrast to CD27, its ligand, CD70, does not appear to be expressed in EMB during periods with or without rejection [12]. This finding, along with the fact that CD70 is preferentially expressed on chronically activated T cells, leads to the speculation that acute rejection is driven rather by an activation of naive T cells than by a reactivation of primed lymphocytes. In contrast to the increased intragraft CD27 mRNA expression, its level in BMC was lower during rejection than during quiescent periods. This finding is in agreement with our studies regarding blood kinetics of mRNA expression of other Tcell activation molecules [21] and our hypothesis to explain this phenomenon is the homing of activated T cells through the blood to the site of rejection [13]. Since soluble CD27 is released by activated T cells, the decline in serum levels of soluble CD27 during acute renal and cardiac rejection, as described in two separate studies [10,11], is in accordance with our findings of decreased CD27 mRNA levels in the blood during rejection. In an allograft recipient, there is a continuous balance between alloimmunity and immunosuppression, the former leading to rejection and the latter allowing the acceptance of the graft. Nevertheless, an alloimmune response seems to
continue even during apparently rejection-free periods. In the present study, we demonstrated that this “subclinical” immune response may be detected by intragraft CD27 mRNA measurements. Indeed, biopsies without any evidence of histological rejection from patients that experienced a rejection (rejectors) showed increased CD27 expression compared to the samples from nonrejectors. No reports on similar markers of risk of rejection have been published, with the exception of the work by De GrootKruseman et al. [22] where high levels of basic fibroblast growth factor mRNA in the first week EMB were associated with acute rejection during the first year following cardiac transplantation. These findings suggest that rejectors present higher levels of alloimmune response even during histologically quiescent periods. Although intragraft CD27 levels alone could not be considered as a marker of rejection risk in the clinical setting, its analysis in combination with other markers, for example basic fibroblast growth factor mRNA, could strengthen the predictive value of molecular diagnosis. In conclusion, the present study has shown that intragraft CD27 gene expression level increases during ongoing rejection and that the serial evaluation of its level during rejection-free periods could be helpful for the discrimination of recipients at high and low risk for presenting acute rejection during the first 6 months after cardiac transplantation. These data are relevant not only to the understanding of some of the mechanisms underlying cardiac allograft acute rejection, but also may contribute to the clinical management of cardiac allograft recipients.
Acknowledgments Supported by Fundac¸ a˜ o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo-FAPESP, and Conselho Nacional de Desenvolvimento Cientifico e Tecnolo´ gico-CNPq. We would like
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to thank Adalberto Socorro da Silva and Elizabeth Cristina P. Hurtado for technical assistance.
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