Antibodies against major histocompatibility complex class I-related chain A in transplant recipients.

Abstract

To review the role of polymorphism of major histocompatibility complex class I-related chain A (MICA) gene and antibodies against MICA antigens in transplant immunology.
The data used in this review were mainly from our own results and from the relevant English language literatures published from 1999 to 2010. Some data presented in this review are in press.
Articles regarding MICA gene discovery and pioneering finding of antibodies against MICA antigen and allograft rejection were selected. This review chronicles the development of our understanding of the role that MICA antigens and antibodies may play in organ transplantation.
Polymorphic glycoprotein MICA antigens were detected on freshly isolated human umbilical cord endothelial cells, but not on peripheral lymphocytes. Antibodies were found and typing of recipients and donors by sequencing the MICA alleles has established that de novo antibodies produced in kidney transplant recipients are directed at mismatched MICA epitopes and are associated with acute rejection and chronic transplant failure. The specificity of antibodies against the epitopes of MICA antigens were well characterized by donor MICA typing, single antigen array testing with antibody absorption and elution. Acute graft-versus-host disease was observed in stem-cell recipients who were mismatched for MICA.
Immunization against mismatched MICA epitopes encountered in donor organs after transplantation may result in antibodies against MICA alleles. Testing for MICA donor-specific antibodies (DSA) which are associated with early failure of kidney transplants may be helpful for identifying some of the targets of antibodies against antigens other than the human leukocyte antigen (HLA) and for improving transplantation outcome.

Full text

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Chin Med J 2011;124(5):764-770
764
Review article

Antibodies against major histocompatibility complex class
I-related chain A in transplant recipients

Yizhou Zou and Peter Stastny

Keywords: major histocompatibility complex class I-related chain A; alloantibody; transplantation

Objective To review the role of polymorphism of major histocompatibility complex class I-related chain A (MICA) gene
and antibodies against MICA antigens in transplant immunology.
Data sources The data used in this review were mainly from our own results and from the relevant English language
literatures published from 1999 to 2010. Some data presented in this review are in press.
Study selection Articles regarding MICA gene discovery and pioneering finding of antibodies against MICA antigen
and allograft rejection were selected. This review chronicles the development of our understanding of the role that MICA
antigens and antibodies may play in organ transplantation.
Results Polymorphic glycoprotein MICA antigens were detected on freshly isolated human umbilical cord endothelial
cells, but not on peripheral lymphocytes. Antibodies were found and typing of recipients and donors by sequencing the
MICA alleles has established that de novo antibodies produced in kidney transplant recipients are directed at mismatched
MICA epitopes and are associated with acute rejection and chronic transplant failure. The specificity of antibodies against
the epitopes of MICA antigens were well characterized by donor MICA typing, single antigen array testing with antibody
absorption and elution. Acute graft-versus-host disease was observed in stem-cell recipients who were mismatched for
MICA.
Conclusions Immunization against mismatched MICA epitopes encountered in donor organs after transplantation may
result in antibodies against MICA alleles. Testing for MICA donor-specific antibodies (DSA) which are associated with
early failure of kidney transplants may be helpful for identifying some of the targets of antibodies against antigens other
than the human leukocyte antigen (HLA) and for improving transplantation outcome.
Chin Med J 2011;124(5):764-770

T
wo groups of investigators were working separately
to describe the major histocompatibility complex
(MHC) class I-related chain (MIC) genes in 1994.
Leelayuwat and his colleagues1 in Perth, Australia named
these genes the PERB11 genes, while another group
working with Spies named them MIC.2 The latter was
selected thus becoming the official name of two
expressed genes, MICA and MICB. They encode proteins
that are similar to human leukocyte antigen (HLA) class 1
gene products but the MIC proteins do not form
tri-molecular complexes due to their lack of association
with beta-2-microtubulin and also do not bind peptides
for presentation to T cells. Instead, the MIC proteins are
ligands for NKG2D3 and are induced during stress.4

EXPRESSION OF MICA ON HUMAN
ENDOTHELIAL CELLS

These antigens are expressed on endothelial cells and are
not found on peripheral blood lymphocytes and both
MICA and MICB are polymorphic. We and others have
observed MICA antigen expression on the surface of
umbilical vein endothelial cells.5,6 Freshly isolated human
umbilical vein endothelial cells were stained with mouse
anti-MICA monoclonal
fluorescence was observed with flow cytometry. But the
staining for MICA was not as strong as compared to HLA
antibody 6B3. Strong
class I staining with the mouse monoclonal antibody
W6/32.

In this work, first polyclonal rabbit antibodies against
MICA were produced by immunizing rabbits with
peptides on constant regions of alpha 1 and alpha 2
domains coupled with keyhole limpet hemocyanin
(KLH).6 It was found that MICA was expressed on
endothelial cells, keratinocytes, and monocytes but not on
CD4+, CD8+ or CD19+
B-lymphocytes from peripheral blood did not stain with
the monoclonal antibody 6B3, which is specific for
MICA. Additionally, freshly isolated human endothelial
cells and the EA.hy926 endothelial cell line were positive
for the anti-MICA antibody staining.

Western blotting and immunoprecipitation detected a
band corresponding to MICA in various cell lines (THP-1,
U937, HeLa, A431, Raji, MOLT-4, and HUVEC),
lymphocytes. T- and
DOI: 10.3760/cma.j.issn.0366-6999.2011.05.024
Transplantation Immunology Division, Department of Internal
Medicine, University of Texas Southwestern Medical Center, USA
(Zou Y and Stastny P)
Correspondence to: Prof. Peter
Immunology Division, Department of Internal Medicine,
University of Texas Southwestern Medical Center, 5323 Harry
Hines Blvd, Dallas Texas 75390-8886, USA (Tel: 1-214-648-3556.
Fax: 1-214-648-2949. Email: Peter.Stastny@UTSouthwestern.edu)
Stastny, Transplantation

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Chinese Medical Journal 2011;124(5):764-770
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Table. MICA gene frequencies in several different ethnic populations (%)
African American NAC Spain South Chinese
8 8 9 10
201 103 154 100

1.2 1.3 7.9 −
28 15.3 16.2 15.5
19 5.5 15.0 0.5
− − 0.7 −
− − − −
0.7 4 1.2 1
32 37.7 25.3 28.5
5.7 8.6 7.1 8.5
0.7 7 4.6 18.5
0.5 2.5 − −
1.2 2.5 − 5
2.7 0 2.5 −
0 1 7.1 −
0.5 2.5 1.2 −
2.5 3.5 2.5 −
1 2.5 − 9.5
− − − −
− − 1.2 −
− − − −
0.5 4.9 2.5 7
− − − −
0.5 − 1.2 −
− − 1.2 −
2.2 − 0.0 −
− − 0.0 4.5
− − 0.7 −
− 0.5 − −
− − − 3.5
− − 0.7 −
− − 0.7 −
− − − 1
NAC: North American Caucasians.
Index
Reference
Total number (2n)
MICA alleles
MICA*001
MICA*002
MICA*004
MICA*005
MICA*006
MICA*007
MICA*008
MICA*009
MICA*010
MICA*011
MICA*012
MICA*015
MICA*016
MICA*017
MICA*018
MICA*019
MICA*020
MICA*023
MICA*026
MICA*027
MICA*029
MICA*030
MICA*033
MICA*041
MICA*045
MICA*046
MICA*047
MICA*049
MICA*050
MICA*052
MICA*Del
North Chinese
11
104

12
4.8

−
−
1.5
23.2
12.5
18.8
0.5
0.1
−
0.1
1.9
0.5
3.8
−
−
−
5.3
−
−
0.5
−
8.7
−
−
−
−
−
3.9
Japanese
12
260

−
14.6
9.2
−
−
1.2
30.8
16.5
10.8
−
12.3
−
−
−
−
3.5
−
−
−
−
−
−
−
−
−
−
−
−
−
−
1.2
Korean
13
278

−
17.6
8.3
−
1.4
4.4
20.1
11.9
19.4
1.1
9.4
−
−
0.4
−
0.4
−
−
−
−
−
−
−
−
3.2
−
−
−
−
−
−
Turkish
14
130

−
15.4
6.2
−
2.3
4.6
12.1
16.9
2.3
3.8
3.1
−
9.2
0.8
7.7
−
3.8
−
−
−
0.8
−
−
−
−
−
−
−
−
−
−
Thai
15
510

−
17.6
3.5
−
−
−
21.4
2.4
18.2
−
3.1
−
0.2
2.2
6.1
15.3
0.4
−
1.4
−
−
−
−
−
−
−
−
−
−
8.2
−


freshly isolated keratinocytes, endothelial cells, and
monocytes but not in CD4+ and CD8+ T cells or CD19+ B
lymphocytes. Upregulation of MICA expression in
different cells by stimulation with gamma-interferon was
not observed, but the expression of MICA was induced in
phytohemagglutinin-stimulated T cells. We confirmed
that MICA was expressed at the cell surface by flow
cytometry. Results of the immunoprecipitation studies of
beta2-microglobulin or MICA-depleted, metabolically
labeled HeLa cells indicated that MICA was not
associated with beta-2-microglobulin.7

SEQUENCING FOR MICA POLYMORPHISMS

The count of alleles is presently at 69 for MICA and 30
for MICB (http://hla.alleles.org/nomenclature/stats.html).
MICA allele frequencies among different populations are
listed in Table from published studies.8-15 A few alleles
make up the majority of the genes found with the rest
being relatively rare (Table). Certainly this polymorphism
is sufficient to consider the possibility that these antigens
might be targets for an allo-immune response in the
setting of organ or stem cell transplantation. The
polymorphism of the MICA alleles is also different from
that of the HLA genes. In the HLA molecules the variable
positions are located prominently in the proximity of the
antigen-binding groove and are heavily concentrated in
exon 2. In the case of the MICA genes, variable positions
are more disperse throughout exons 2 and 3 and generally
only two alternative amino acids can be found for any
given position.

Another feature of the polymorphism of MICA is the
variation in the trans-membrane (TM) region. Several
alleles have identical extracellular domains and differ
only in the TM region. This region consists of a variable
number of GCT repeats. Therefore typing for the
polymorphism in the TM region is essential to avoid
typing ambiguities. We have developed a procedure that
allows identification of the TM polymorphisms, based on
the sequencing signals without any additional assays.16
This is the method of choice for high resolution typing of
the MICA alleles and it is what we use in our day-to-day
work.

FIRST ATTEMPTS TO TEST FOR
ANTIBODIES AGAINST MICA

In 1999, after having gained evidence suggesting that
MICA was expressed in endothelial cells, keratinocytes
and monocytes, but not in CD4+, CD8+ or CD19+
lymphocytes, we decided to attempt to produce
recombinant MICA antigens in order to test the
possibility that antibodies against MICA may be

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produced in humans. This work was performed by
Norberto Zwirner and was published the following year.17
To analyze whether MICA may be a target for specific
antibodies in sera of transplanted patients, we produced
three recombinant MICA proteins consisting of the alpha
1, alpha 2, and alpha 3 domains, and used them in an
enzyme-linked immunosorbent assay.

ANTIBODIES AGAINST MICA ALLELES

We found that several patients had specific antibodies
against MICA. Most of them were detected in serum
samples collected at different times after organ rejection.
Although this finding raises the question of how these
patients became immunized, the fact that the polymorphic,
HLA-like MICA molecule, expressed at the cell surface
of endothelial cells, is recognized by specific antibodies
in sera of transplanted patients, suggests that MICA may
be a target molecule in allograft rejection.17

RECOMBINANT MICA PROTEINS AND OTHER
METHODS FOR AN IMMUNOASSAY TO DETECT
ANTIBODIES

In order to develop an assay for antibodies against MICA
antigens with Luminex beads, we produced recombinant
MICA antigens in insect cells. These constructs contained
the signal peptide and the extracellular domains of
MICA*001, MICA*002, MICA*004, MICA*008 and
MICA*009. They also contained a six-his-tag sequence
and a biotinylation peptide. The proteins were expressed
in HighFive insect cells, were purified with nickel affinity
agarose and attached to Luminex beads.18 In the course of
subsequent work, several modifications have been made
to the preparation of Luminex-coupled MICA reagents
produced in our laboratory. The constructs were changed
and the biotinylation peptide sequence was omitted; the
insect cells used were Sf9 cells instead of the HighFive
cells; and the conjugation of the MICA proteins to
carboxylated Luminex beads was performed using
carbodiimide hydrochloride. Also, two steps of
purification of the recombinant proteins were used. The
first is based on the his-tag using nickel agarose, the
second was immuno-absorption
monoclonal antibody 6B3, attached to sepharose beads.
These steps have virtually eliminated non-specific
reactions and the resulting assay detects true antibodies to
MICA proteins which can be validated by absorption and
elution procedures.19

REJECTION OF KIDNEY:
MICA ANTIBODIES IN ELUATES

Zou et al18 kindly provided 59 eluates obtained from
specimens of kidney
immunological rejection. In this group of samples there
were 14 from transplants lost due to hyperacute rejection,
15 with acute rejection and 30 were from kidneys lost due
to chronic rejection. Many of these recipients had
with anti-MICA
transplants undergoing
received previous transplants by which they could have
been immunized. Antibodies against MICA were detected
in 11 of these specimens. Antibodies against HLA
antigens were recovered in some of the same eluates. In 6
eluates both MICA and HLA antibodies were found; in 17
we found only HLA antibodies; and in 5 eluates only
antibodies against MICA antigens were detectable. These
results suggested that MICA antibodies may have been
involved in kidney transplant rejection.

DECREASED KIDNEY ALLOGRAFT SURVIVAL
IN PATIENTS WITH PRE-TRANSPLANT
ANTIBODIES AGAINST MICA

Applying the materials of the Collaborative Transplant
Study (CTS), we retrospectively analyzed a large number
of kidney transplant patients for whom the outcome after
transplantation was already known. A blind study was
carried out with sera shipped to our laboratory. The
presence of antibodies against MICA in serum obtained
prior to transplantation correlated with an increased
frequency of graft loss in our results. This was
especially true in recipients who had received kidneys
that were well matched for HLA.20 Nineteen hundred and
ten kidney transplant recipients were studied to determine
IgG antibodies against
MICA*004, MICA*008 and MICA*009. We used a
method that was developed in our laboratory where we
bound antibodies to recombinant MICA antigens bound
to Luminex beads. After transplantation, allograft
function was analyzed at 3, 6 and 12 months. Using
Long-rank analysis, graft survival was compared in
patients with and without antibodies against MICA
antigens. A multi-factorial Cox regression analysis was
also performed. It was not determined whether the
antibodies were reactive with the MICA antigens of the
donors because donor DNA was not available for study.
Of the 1910 patients studied, 217 patients (11.4%) were
found to have antibodies against MICA. The MICA
antibodies that were found were associated with an
increase in kidney-allograft failure, possibly due to
rejection. Recipients who were well-matched (0 or 1
HLA-A plus HLA-B plus HLA-DR) and patients without
antibodies against HLA antigens (PRA=0) showed
especially evident association between the presence of
antibodies against MICA and early graft loss.

POLYMORPHISMS OF MICA RECOGNIZED BY
HUMAN ANTIBODIES

Eleven selected common MICA antigens were coupled to
polystyrene beads using the technique described above. In
addition, two hybrid MICA molecules and two
preparations produced by site-directed mutagenesis that
have single-amino acid substitutions were used.
HMy2.CIR cells were transfected with MICA*001, *002,
*008 and *009. The basic procedure was to first
determine the pattern of a serum on MICA-conjugated
Luminex beads, then absorbed the serum with transfected
cell expressing only one defined MICA allele, and finally
MICA*001, MICA*002,

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Figure 1. Indentfication of MICA epitopes by antibody absorption and elution. Serum selected containing MICA specific antibodies were
detected by single antigen Luminex bead assay before absorption by MICA*008 transfected cells. Antibody detection in absorbed serum
after absorption and in elution from absorption is shown. A: before absorption. B: after absorption. C: elution from absorption.

eluted the antibodies by treatment with acid. The
absorbed serum and the eluate were both back-tested on
the Luminex bead set. This procedure is described
previously.19 The effects on antibody of absorption and
elution were demonstrated as shown in Figure 1. A serum
containing antibodies against MICA*001, MICA*008
and MICA*009, but not MICA*002 was absorbed by
MICA*008 transfected cells. The antibodies against
MICA*008 and MICA*009 was removed, but antibodies
against MICA*001 remained after absorption. Absorbed
antibodies could be recovered by elution from the
absorbed cells and detected by Luminex beads again.
Fourteen patterns of reactivity were identified19 from this
experiment, including antibodies recognizing single
alleles, small groups consisting of 2, 3, or 4 alleles, and
some larger, more complex groups called MICA-G1 and
MICA-G2. The definition of target epitopes relates to
donor-recipient MICA mismatches and supports the
hypothesis that these antibodies are for the most part, the
results of donor-specific allo-immunization. Using mainly
the analysis of epitopes by computer,21 similar results
were obtained by Duquesnoy and coworkers. Analysis of
the reactivity of 6 sera from transplant recipient using a
commercial kit of recombinant MICA proteins bound to
Luminex beads showed patterns which were found to be
in agreement with the computer analysis.21

ANTIBODIES DEVELOPING IN KIDNEY
TRANSPLANT RECIPIENTS AFTER
TRANSPLANTATION ARE DONOR SPECIFIC

A large pool of kidney transplant recipients and donors
were studied to determine the donor-specificity of
antibodies against MICA in a collaborative study with
Steve Cox and co-workers (unpublished data). Using
sequence-based typing, over 200 patients and their donors
were typed for MICA alleles and MICA antibodies. Three
available MICA conjugated Luminex beads kits were
used, of which two resulted in positive results. After
transplantation, most of the antibodies that developed
were specific for mismatched MICA alleles present in the
donor. These antibodies were associated with acute
rejection determined from transplant biopsies. They were
also often associated with decreased graft function and
evidence of chronic graft damage. Detailed analysis of
mismatched MICA epitopes showed that antibody
development was correlated with MICA residue
mismatching. Therefore, while 94% of patients with
MICA residue mismatch developed antibodies against
MICA, antibodies were not produced when the MICA
residues were matched. These patterns of reactions
corresponded to patterns of antibodies against MICA
epitopes we previously reported.19 Our findings conclude
that mismatching for MICA alleles may lead to
production of antibodies against MICA, usually in the
first year of the transplant, and that strong antibodies
against MICA were often associated with acute rejection.

FREQUENCY OF ANTIBODIES AGAINST MICA
IN ORGAN TRANSPLANT RECIPIENTS

Current reagents are available to detect true MICA
antibodies that consistently correspond to mismatched
alleles of the immunizing donors. These kind of
antibodies can be removed through absorption with cells
transfected with specific MICA alleles. The eluates from
the absorbed cells can also reproduce the same serologic
patterns as obtained with the original serum.19 In patients
who have not received transplantation, such antibodies
are seen in only about 10% of patients waiting for their
first kidney transplantation. In the post-transplant period,
we observed an increase in antibodies to MICA antigens.
Such antibodies are donor-specific and correspond to
mismatched MICA epitopes, which correlate with
rejection. Approximately 20% of kidney transplant
recipients have anti-MICA antibodies in our experience.
When the donor and the recipient MICA epitopes were
matched, these antibodies did not develop. In patients
who have rejected a previous transplant, we also found
further increase in the frequency of antibodies against
MICA. This group of recipients makes up approximately
30%, and may have antibodies against MICA alleles.

ANTIBODIES AGAINST DONOR MICA ARE
ASSOCIATED WITH LOSS OF KIDNEY
ALLOGRAFT FUNCTION

Various investigators have been studying the role of
antibodies against MICA alleles on the post-transplant
course of renal transplants.18,20,22-24 In most cases, it is

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hard to establish the role antibodies against MICA when
HLA antibodies were also present. In spite of this, reports
show that many patients without HLA antibodies detected
by Luminex assay have kidney allograft failure associated
with antibodies to MICA. Our recent analysis with Steve
Cox and colleagues concludes that antibodies against
MICA were specific for the MICA alleles of the donor
and is strongly associated with failure of the grafts
(unpublished data).

ANTIBODIES AGAINST MICA REPORTED TO BE
ASSOCIATED WITH REJECTION OF HEART
AND LUNG TRANSPLANTS

A study published by Suarez-Alvarez and coworkers25
associates antibodies against MICA antigens with acute
episodes of rejection in heart transplant recipients.
Therefore, it is likely that these antibodies may also play
a role in heart transplants. Moreover, recent reports
suggest that antibodies against MICA antigens were
associated with chronic rejection of lung transplants.26 In
our group, we found that HLA/MICA alloantibodies
associated with the development of coronary artery
disease (CAD) after heart transplantation. Eighty percent
of recipients without antibodies were free from of CAD.
Fifty-six percent of recipients with MICA antibodies
alone and 30% of recipients who have MICA antibodies
and HLA antibodies were free from CAD. The data
suggested that patients with both MICA and HLA
antibodies may be at very high risk to develop CAD
(Figure 2).



Figure 2. Percent of CAD free among different patient groups
with or without MICA antibodies.

VIRTUAL CROSS-MATCHING FOR MICA

Pre-immunization against mismatched MICA epitopes
encountered in donor organs after transplantation may
result in antibodies against MICA allele. Testing for
MICA donor-specific antibody (DSA) which appear to be
linked with early failure of kidney transplants may be
helpful for improving post transplantation management.
Since patients who have rejected a MICA mismatched
allograft have a high likelihood to developed antibodies
against MICA, it is important to characterize the
specificity of the antibodies in the recipient’s serum and
to determine the MICA alleles of prospective donors.
This “virtual crossmatch” may be useful for avoiding the
loss of allografts against which patients are already
sensitized because conventional T- and B-lymphocyte
cross- matching applied for HLA antibody does not work
with MICA antibodies. To present, there has not been a
well-documented case of hyperacute rejection due to
antibodies against MICA. Steve Cox and coworkers
(unpublished data) had an interesting finding that all of
kidney biopsies they tested demonstrated the negative
C4d staining result. These kidney samples were stained
by the immuno-peroxidase method which is somewhat
less sensitive than immunofluorescence. C4d staining of
the peritubular capillaries is not necessary to detect
antibody-mediated rejection due to HLA antibodies. Both
humoral and cellular mechanisms play a role in most
cases of rejection.

ASSOCIATION OF MISMATCHING FOR MICA
WITH ACUTE GRAFT-VERSUS-HOST DISEASE
IN WELL MATCHED STEM CELL RECIPIENTS

It was hypothesized that MICA can be recognized as
transplantation antigens and that they might have an
effect in stem cell transplantation. Based upon this
consideration, we then proceeded to conduct a study of
HLA well-matched stem-cell transplant patients at MD
Anderson Hospital in Houston. MICA was mismatched in
only 8.4% while the majority for HLA-A, B, C, DR and
DQ was matched in 73% (10/10 matches). Many of the
HLA-matched pairs were also matched for MICA27 due
to the linkage disequilibrium between MICA and HLA. In
MICA mismatched patients, a higher rate of grades II–IV
acute graft-versus-host-disease was observed (80% vs.
40%, P=0.003). For MICA mismatched patients, higher
occurrence of gastrointestinal acute graft-versus-host
disease was also seen. These findings lead to the
conclusion that MICA
transplantation antigens possibly recognized by human T
cells. Previously, we observed that T cells from mice
immunized with recombinant MICA proliferated in
response to MICA and that these cytotoxic T cells which
developed could kill monocytes pulsed with MICA.28

CONCLUSION

The MICA antigens are absent from peripheral blood
lymphocytes but are expressed on endothelial cells, they
are polymorphic and can induce an immune response in
transplant recipients, consisting of formation of
antibodies, against mismatched epitopes. Early studies by
our group indicated that antibodies against MICA
antigens were associated with decreased survival of
kidney transplants. More recent work has established that
de novo antibodies against MICA are produced by
may represent novel