Post-transplant soluble MICA and MICA antibodies predict subsequent heart graft outcome

Transplant Immunology 17 (2006) 43 – 46 www.elsevier.com/locate/trim

Post-transplant soluble MICA and MICA antibodies predict subsequent heart graft outcome Beatriz Suárez-Álvarez a , Antonio López-Vázquez a , Roberto Díaz-Peña a , Beatriz Díaz-Molina b , Rosa M. Blanco-García c , M. Rocío Álvarez-López c , Carlos López-Larrea a,⁎ a

Histocompatibility and Transplantation Unit, Hospital Universitario Central de Asturias, 33006-Oviedo, Spain b Department of Cardiology, Hospital Universitario Central de Asturias, Oviedo, Spain c Department of Immunology, Hospital Virgen de la Arrixaca, Murcia, Spain Received 30 August 2006; accepted 13 September 2006

Abstract The objective of this retrospective study was to evaluate the role of MICA in heart graft acceptance. Pre- and post-transplant sera from 31 patients were evaluated for MICA antibodies by cytotoxicity on recombinant cell lines and soluble MICA (sMICA) concentrations by ELISA. The results demonstrated that the patients with post-transplant anti-MICA antibodies were at a high risk for the development of severe acute rejection (AR) ( p b 0.03; OR = 8.5). However, the presence of post-transplant sMICA was found to be associated with functioning grafts without AR episodes ( p b 0.03, OR = 7.9). In this preliminary survey, the negative association of sMICA with AR was found to be in the absence of MICA antibodies. Further research is needed to clarify the role of sMICA in allograft acceptance. Post-transplant evaluation of humoral immune response to MICA and the measure of sMICA in patient's sera may provide a good predictor of AR. © 2006 Elsevier B.V. All rights reserved. Keywords: MICA antibodies; Soluble MICA; Heart transplantation; Rejection

Alloantibodies involved in transplantation are directed against HLA-class I and class II molecules. However, other antibodies against molecules such as MICA expressed on endothelial cells have been associated with graft loss. This molecule (MICA) shows homology with classical HLA-class I but has no role in antigen presentation. This is a highly polymorphic cell surface glycoprotein mainly expressed on endothelial, epithelial cells, fibroblasts and activated monocytes [1]. The expression of MICA is induced by stress situations and is up-regulated during infection and tumour transformation [2,3]. This protein is a ligand for NK and CD8+ T cells, which express NKG2D, a common activating natural killer cell receptor [4]. The NKG2D receptor acts as an activating

Abbreviations: HLA, Human Leukocyte Antigens; MICA, MHC class I chain-related molecule A; NK, natural killer cells; AR, acute rejection. ⁎ Corresponding author. Tel.: +34 985 10 61 30; fax: +34 985 10 61 95. E-mail address: [email protected] (C. López-Larrea). 0966-3274/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2006.09.014

immunoreceptor in NK cells and as a co-stimulatory signal in CD8+ T cells which complements TCR-mediated antigen recognition on target cells [5]. Recently, several papers have been focused on MHC class Irelated MIC genes products as possible candidates for treatment during transplantation course. The finding that MICA is surfaceexpressed on endothelial cells makes this polymorphic molecule a possible target for both humoral and cellular immune responses during graft rejection. In fact, renal and pancreatic grafts with evidence of both acute and chronic rejection have been shown to express MIC proteins [6,7], and anti-MIC antibodies have been identified in the serum of patients [8]. It has also been reported that soluble MICA (sMICA) is released from the cell surface of tumour cells and can be detected in the sera of these patients [9–12]. This soluble form engages cells expressing NKG2D, induces endocytosis and degradation of this receptor and impairs responsiveness to tumour cytolysis [13]. Shedding of MICA by tumour cells may modulate NKG2D-mediated antitumour immune surveillance. These results support the possible

44

B. Suárez-Álvarez et al. / Transplant Immunology 17 (2006) 43–46

Table 1 Clinical characteristics of heart transplanted patients Characteristic

Number of patients n = 31 (%)

Age (years) Gender (male/female) Previous heart disease Dilated cardiomyopathy Coronary disease Valvulopathy Induction treatment Zenapax Simulec None Immunomodulatory treatment CsA + MMF FK506 + MMF Age donor (years) Gender donor (male/female) Rejection (1st year) Yes No

52 ± 10 27 (87.1%)/4 (12.9%) 18 (58%) 12 (38.7%) 1 (3.3%) 8 (25.8%) 6 (19.4%) 17 (54.8%) 28 (90.3%) 3 (9.7%) 35 ± 11 20 (64.5%)/11 (35.5%) 8 (25.8%) 23 (74.2%)

CsA: Cyclosporine; MMF: Mycophenolate.

involvement of sMICA in the regulatory mechanisms that occur during human allotransplantation. In order to investigate the contribution of MHC-class I nonclassical MICA in graft tolerance, we analyzed the presence of soluble MICA levels and the development of anti-MICA antibodies in the serum of patients after heart transplantation and these correlated with the incidence of acute rejection. This study consisted of 31 patients (27 men and 4 women, mean age 52 ± 10) all of whom had received a heart transplant and 20 healthy donors (42 ± 10) were used as controls. Heart transplantations were performed between 2000 and 2003 in two Spanish Hospitals (Hospital Universitario Central de Asturias and “Virgen de la Arrixaca”). The study was approved by the Ethics Committees of our hospitals and all patients gave written informed consent. Each patient had one serum sample taken pre-transplant and an average of 2.4 samples post-transplant. A complete screening for HLA antibodies was carried out for all patients previous to transplantation, and none of them were positive for these antibodies. Clinical features are shown in Table 1. The degree of acute rejection was established on the basis of clinical and histopathological data, and was classified according to the criteria of the International Society of Heart and Lung Transplantation (ISHLT). The patients were classified into two groups: (1) with rejection (R), comprising 8 patients who developed at least one episode of severe acute rejection (histological grade ≥ 3A) during the first year after grafting, and (2) with functioning graft and non-rejection (NR), comprising 23 patients with grades b 3A. Blood samples were obtained at different times (at fifteen days, three months and one year post-transplantation), sera were collected and frozen at − 80 °C until further analysis. A human MICA ELISA kit (IMMATICS Biotechnologies, Germany) was used to detect soluble MICA in sera from HTX patients and healthy controls, following the manufacturer's

protocol. The absorbance was measured at 492 nm and the sensitivity was 100 pg/ml. The results shown are the means of triplicates. Patients who had a serum concentration of sMICA greater than 400 pg/ml were considered sMICA positive. HeLa and several B cell lines (DUCAF, BM15, JESTHOM, LWAGS and BM16) obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were selected to express the alleles MICA⁎008, ⁎001, ⁎004, ⁎007, ⁎011 and ⁎018 respectively. The MICA⁎002 allele was generated by sitedirected mutagenesis using MICA⁎007 cDNA as the template. Full-length MICA cDNA was amplified using the following primers: 5′-TCTGGATCCATGGGGCTGGGCCCG 3′ (sense) and 5′-CACGAATTCCTAGGCGCCCTCAGTGGAG 3′ (antisense). The product was inserted into BamHI/EcoRI sites in the plasmid pBluescript II-KS+ (Stratagene, San Diego, CA), and thereafter shuttled to Not I/Xho I sites in the pREP-4 plasmid (Invitrogen, Carlsbad, CA, USA). E. coli DH10B bacteria were transformed with ligation mixture and one clone containing a MICA insert, as verified by sequencing, was selected. An MHC class I cell surface negative human B-lymphoblastoid cell line (HMy2.C1R) was transfected by electroporation with different MICA alleles using standard procedures. Stable transfectant cells were grown in RPMI 1640 ± 10% heat inactivated FCS and selected with Hygromycin B at 800 μg/ml. Surface expression of different MICA alleles was analyzed in human recombinant cell lines by flow cytometry with AMO-1 MAb. All were found to express MICA on the surface (data not shown). The complement-dependent cytotoxicity test was used to detect the specific antibody against MICA. We used seven MICA antigen expressing human recombinant cell lines: MICA 001, 002, 004, 007, 008, 011, and 018. The HMy2.CIR cell line was tested as controls. The complement-dependent cytotoxicity test was performed following the standard protocols. A specific dead cell count of more than 50% was considered as positive for MICA antibodies. Descriptive data are presented as mean ± standard deviation. Significance between frequencies was determined by Fisher's exact test. The probability factor b0.05 was considered significant. During the first year post-transplant, 8 patients (25.8%) experienced AR whilst 23 (74.2%) had functioning grafts without rejection. Soluble MICA levels were detected between days 15 and 20 post-graft implantation in the serum of 19 (61.3%) of the 31 patients analysed and were undetectable in the serum of 12 patients (38.7%) (Fig. 1A). Clinical analysis showed that 17 out of 23 patients (73.9%) that did not develop severe acute rejection episodes during the first year, had detectable sMICA ( p b 0.03; OR = 8.5). In contrast, only two of the rejected patients were in the sMICA positive group. The correlation between the presence of sMICA and the absence of rejection was also maintained in 80% of patients during at least the three different times tested. Sera of the 20 healthy volunteers contained only low levels of sMICA close to the detection limit of the ELISA (data not shown). Therefore, soluble MICA levels may modulate allograft responses and stable graft function. Only sera from two patients contained HLA-class I antibodies post-transplant. Anti-MICA allele-specific antibodies were detected by cytotoxicity assay using the MICA antigen expressing

B. Suárez-Álvarez et al. / Transplant Immunology 17 (2006) 43–46

45

Fig. 1. Distribution of soluble MICA and MICA antibodies in heart transplanted patients. sMICA was detected in patients who did not develop severe acute rejection episodes during the first year post-transplant ( p b 0.03) (1A) whilst the presence of anti-MICA antibodies was higher in patients with acute rejection ( p b 0.03) (1B). Combined analysis of sMICA and anti-MICA antibodies in heart transplant patients during the first year post-transplant (1C).

cell line HMy2.C1R. Of the 31 recipients studied, 9 (29%) produced antibodies against MICA (Fig. 1B). The presence of MICA Abs was significantly higher in patients with AR (62.5%) than in the group of non-rejected patients (17.4%; p b 0.03; OR = 7.9). Pre-transplant anti-MICA antibodies were only detected in two patients who were not associated with AR. However, among the nine post-transplant patient's sera with anti-MICA antibodies, five presented at least one episode of AR during the first year after transplantation. We determined whether the combined development of antiMICA antibodies and the presence of sMICA influence the outcome of the transplant (Fig. 1C). It was difficult to analyse the clinical significance of the multifactorial analysis due to the size of the population. However, we found some tendency for antiMICA Ab(+) to occur in the absence of sMICA in patients with AR (37.5% vs. 0% in patients that did not develop episodes of AR). Conversely, we found that the presence of sMICA and the absence of anti-MICA Ab(−) was found in 56.5% of patients who had a transplant without acute rejection and was absent in patients with AR. These preliminary results need to be confirmed, but suggest that patients having sMICA would show better graft acceptance in the absence of MICA antibodies. Although several studies have found the presence of antiHLA Abs in heart transplant recipients associated with rejection, an important group of patients who rejected did not have detectable HLA antibodies. In addition, there are considerable data indicating that antibodies to non-HLA antigens, such as endothelial molecules, can contribute to acute antibodymediated cardiac rejection. As candidates, MICA and MICB are of particular interest because these polymorphic antigens are detected on endothelial cells but not lymphocytes. MICA antibodies have previously been implicated with acute renal

allograft rejection and loss [7]. In this retrospective study, we compared the accuracy of a panel of reactive antibodies antiMICA with the presence of sMICA in predicting acute rejection episodes post-heart transplant. The development of the CDC assay on HMy2.C1R transfected cell lines, has made the detection of anti-MICA Abs possible. Patients positive for MICA antibodies were at significantly higher risk of acute rejection ( p b 0.03, OR = 7.9). Our results demonstrate the prognostic value of anti-MICA antibodies for predicting the development of AR. Renal and pancreatic grafts with evidence of both acute and chronic rejection have been shown to express MIC proteins [6]. It is important to determine the histopathologic expression of MICA in heart grafts with acute rejection. In contrast with the presence of MICA antibodies associated with AR, we found an inverse relationship between sMICA levels and recurrent severe rejection ( p b 0.03; OR = 8.5). Preliminarily, we found that this association was found in patients that did not develop anti-MICA antibodies. In addition, the association of MICA antibodies to AR was found in the absence of sMICA. More extensive study needs to be done to definitively determine whether the interplay between the presence of sMICA and anti-MICA antibodies may influence the frequency of acute rejection. It has been described that the expression of the non-classical molecule HLA-G in biopsies and in the sera of patients who have undergone heart and renal transplantation is associated with a better graft tolerance [14,15]. The tolerogenic properties of HLA-G act via specific inhibitory receptors present on immunocompetent cells. We can speculate that the presence of sMICA may act to inhibit the humoral response against MICA, thus inhibiting B cell function or suppressing the efficiency of anti-MICA recognition. Additionally, sMICA expression may

46

B. Suárez-Álvarez et al. / Transplant Immunology 17 (2006) 43–46

also play a role in cellular rejection. Soluble MICA, by interacting with NKG2D, may down modulate the receptor on T cells and NK cells, rendering the cells unresponsive. Therefore, sMICA may induce inhibition of CD8+ T and NK cell functions and may participate in allograft tolerance. In conclusion, this preliminary study suggests that the measurements of sMICA and MICA antibodies can be of prognostic value in the assessment of patients after heart transplantation. Acknowledgement This work was supported by the Spanish grants: FICYT PC04-37, “Mútua Madrileña 2005–2007” and FIS (RED G03/03 and RED G03/104). References [1] Bahram S, Bresnahan M, Geraghty DE, Spies T. A second lineage of mammalian major histocompatibility complex class I genes. Proc Natl Acad Sci U S A 1994;91:6259–63. [2] Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T. Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB. Proc Natl Acad Sci 1999;96:6879–84. [3] Groh V, Bruhl A, El-Gabalawy H, Nelson JL, Spies T. Stimulation of T cell autoreactivity by anomalous expression of NKG2D and its MIC ligands in rheumatoid arthritis. Proc Nat Acad Sci U S A 2003;100:9452–7. [4] Wu J, Song Y, Bakker AB, et al. An activating immunoreceptor complex formed by NKG2D and DAP10. Science 1999;285:730–2.

[5] Bauer S, Groh V, Wu J, et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 1999;285:727–9. [6] Hankey KG, Drachenberg CB, Papadimitriou JC, et al. MIC expression in renal and pancreatic allografts. Transplant 2002;73(2):304–6. [7] Quiroga I, Salio M, Koo DD, Cerundolo L, Shepherd D, Cerundolo V, et al. Expression of MHC class I-related Chain B (MICB) molecules on renal transplant biopsies. Transplant 2006;81(8):1196–203. [8] Zwirner NW, Marcos CY, Mirbaha F, Zou Y, Stastny P. Identification of MICA as a new polymorphic alloantigen recognized by antibodies in sera of organ transplant recipients. Hum Immunol 2000;61:917. [9] Salih HR, Antropius H, Gieseke F, et al. Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood 2003;120(4):1389–96. [10] Wu JD, Higgins LM, Steinle A, Cosman D, Haugk K, Plymate SR. Prevalent expression of the immunostimulatory MHC class I chain-related molecule is counteracted by shedding in prostate cancer. J Clin Invest 2004;114(4):560–8. [11] Raffaghello L, Prigione I, Airoldi I, et al. Downregulation and/or release of NKG2D ligands as immune evasion strategy of human neuroblastoma. Neoplasia 2004;6(5):558–68. [12] Jinushi M, Takehara T, Tatsumi T, et al. Impairment of natural killer cell and dendritic cell functions by the soluble form of MHC class I-related chain A in advanced human hepatocellular carcinomas. J Hepatol 2005;43 (6):1013–20. [13] Groh V, Wu J, Yee C, Spies T. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature 2002;419(6908):734–8. [14] Lila N, Amrein C, Guillemain R, et al. Human leukocyte antigen-G expression after heart transplantation is associated with a reduced incidence of rejection. Circulation 2002;105:1949–54. [15] Qiu J, Terasaki PI, Miller J, Mizutani K, Cai J, Carosella ED. Soluble HLA-G expression and renal graft acceptance. Am J Transplant 2006 [Electronic publication ahead of print].