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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) ( pb0.03; OR =8.5). However, the presence of post-transplant sMICA was found to be associated with functioning grafts without AR
episodes (pb0.03, 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
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 I-
related MIC genes products as possible candidates for treatment
during transplantation course. The finding that MICA is surface-
expressed on endothelial cells makes this polymorphic molecule
a possible target for both humoral and cellular immune res-
ponses 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 anti-
tumour immune surveillance. These results support the possible
Transplant Immunology 17 (2006) 43–46
www.elsevier.com/locate/trim
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: inmuno@hca.es (C. López-Larrea).
0966-3274/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.trim.2006.09.014
involvement of sMICA in the regulatory mechanisms that occur
during human allotransplantation.
In order to investigate the contribution of MHC-class I non-
classical 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 Trans-
plantation (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 b3A.
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 site-
directed 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′(anti-
sense). 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 posi-
tive 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 ( pb0.03; 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 de-
tected by cytotoxicity assay using the MICA antigen expressing
Table 1
Clinical characteristics of heart transplanted patients
Characteristic Number of patients
n=31 (%)
Age (years) 52±10
Gender (male/female) 27 (87.1%)/4 (12.9%)
Previous heart disease
Dilated cardiomyopathy 18 (58%)
Coronary disease 12 (38.7%)
Valvulopathy 1 (3.3%)
Induction treatment
Zenapax 8 (25.8%)
Simulec 6 (19.4%)
None 17 (54.8%)
Immunomodulatory treatment
CsA+ MMF 28 (90.3%)
FK506+MMF 3 (9.7%)
Age donor (years) 35±11
Gender donor (male/female) 20 (64.5%)/11 (35.5%)
Rejection (1st year)
Yes 8 (25.8%)
No 23 (74.2%)
CsA: Cyclosporine; MMF: Mycophenolate.
44 B. Suárez-Álvarez et al. / Transplant Immunology 17 (2006) 43–46
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%; pb0.03;
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 anti-
MICA 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 anti-
MICA 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 anti-
HLA 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 antibody-
mediated 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 anti-
MICA 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 ( pb0.03, 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 ( pb0.03; 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
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 ( pb0.03) (1A) whilst the presence of anti-MICA antibodies was higher in patients with acute rejection ( pb0.03) (1B).
Combined analysis of sMICA and anti-MICA antibodies in heart transplant patients during the first year post-transplant (1C).
45B. 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 PC-
04-37, “Mútua Madrileña 2005–2007”and FIS (RED G03/03
and RED G03/104).
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