4. General discussion 4.1 Minor antigens in renal transplantation Several years ago, a possible ...

64 Pages·2012·0.57 MB·English

4. General discussion
4.1 Minor antigens in renal transplantation
Several years ago, a possible role of non-HLA immunity in allograft injury has
been suggested after data from The Collaborative Transplant Study showed an
unexpected negative effect of PRA on graft survival in HLA-identical sibling
transplants (182). The effect of PRA was apparent only beyond one year post-
transplantation and progressed slowly over time in that population in comparison
with deceased donor patients who were subjected to earlier graft loss. A possible
explanation was that antibodies to minor histocompatibility antigens occurred
frequently with anti-HLA antibodies or cross-reacted with epitopes shared by HLA
antibodies. However, the finding that a substantial proportion of sibling
transplants failed even in the absence of any detectable antibody reactivity
suggested that non-HLA immunity could contribute significantly to graft loss.
Antibodies to non-HLA antigens have been pointed out as likely to be involved in
renal allograft rejection and graft loss throughout associative studies. The most
abundant literature in that field undoubtedly relates to MICA antibodies followed
by antibodies to AT1R and antibodies to vimentin or agrin. However, no
experimental model was able to demonstrate at the present time a real
pathogenicity of those antibodies, especially as a substantial proportion of them
has been reported to be auto-antibodies and therefore, might be simple artefacts
due to cross-reactivity with epitopes from infectious agents for example.
Since MICA had appeared in recent years as a non-HLA antigenic target in renal
transplantation, we decided to start a research in 2008 because little was actually
known in that field at that time, especially with regard to epidemiology, risk
factors for sensitization and the autologuous or allogeneic nature of MICA
antibodies. By a happy coincidence, we received support from a firm that aimed
to market a new kit for identifying MICA antibodies by Luminex technology.
4.2 Epidemiology of MICA antibodies and risk factors for MICA sensitization
Our work showed that MICA sensitization was 4-fold higher among CKD patients
than in healthy volunteers. Unexpectedly, the prevalence of MICA antibodies was
also found to be 2 fold higher among males as compared to females while we
could identify pregnancy as an independent risk factor for MICA sensitization.
The origin of the influence of male gender on the presence of MICA antibodies
has not been investigated yet but mechanisms controlling MICA expression
might be related to sex. The other routes for MICA allo-immunisation were mainly
those involved in HLA immunization: previous graft and blood transfusions. In
addition, uremia “per se” also appeared as an independent risk factor for the
development of MICA antibodies. Nevertheless, the presence of MICA antibodies
among controls suggested that other possible routes for MICA sensitization might
exist. A possible explanation might be cross-reactivity against epitopes shared by
for example, both microbial agents and MICA antigens.
Since MICA is a cell-surface stress-inducible glycoprotein expressed on ECs, the
high prevalence of MICA antibodies among CKD patients might be related to the
“chronic microinflammatory state” of such patients. This “microinflammatory
state” relates to an increased percentage of CD14+/CD16+ monocytes in
peripheral blood that promote endothelial injury through the release of pro-
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inflammatory cytokines and to patient-related factors, such as underlying
disease, comorbidity, oxidative stress, infectious, genetic and immunologic
factors, as well as those arising from dialysis treatment itself, mainly membrane
and dialysate biocompatibility (317). Indeed, chronically haemodialyzed patients
were recently shown to exhibit a profound decrease in NKG2D bearing cells
within both the CD8+ T cell and NK cell populations, and conversely, both
increased membrane-bound MICA on monocytes and soluble MICA levels,
possibly leading to the induction of MICA antibodies (318).
4.3 Impact of MICA antibodies on graft survival
Since 5 years approximately, several groups reported an association of MICA
antibodies with lower graft survival or rejection (290, 291, 300, 304). However,
those previous publications showed a crude univariate analysis of the
relationship between MICA antibodies and graft outcomes without investigating
the impact of possible confounding factors likely to influence graft loss. Until now,
no study in renal transplantation gave the formal proof of neither the
pathogenicity of such antibodies nor that those possibly deleterious antibodies
were DSA. Moreover, positive MICA reactivity had not been confirmed on MICA
expressing cell lines. Up to now, 2 studies tried to demonstrate the donor
specificity of MICA antibodies. The first was performed in heart transplant
recipients and it concluded to a lack of a deleterious impact of MICA antibodies
whether they were DSA or NDSA (294). The second aimed to address the effect
of MICA mismatching on the development of MICA antibodies but did not focus
on graft loss (306).
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To date, how MICA antigens might be deleterious to the graft remains unclearly
understood. Especially, the possibility that MICA and HLA antibodies could act in
concert to allograft injury is not determined.
Unlike Zou’s and Terasaki’s studies, our findings showed a clear lack of effect of
either pre- or post-transplant MICA antibodies on renal graft outcomes. This
discrepancy might be explained by differences in immunosuppressive regimen
given to patients. In our 2 studies, patients received a higher overall amount of
immunosuppression, probably modulating the allo-immune response to the graft
and therefore, benefiting graft outcome. Indeed, our patients received more
frequently tacrolimus and MPA as maintenance therapy rather than cyclosporine
and azathioprine in Zou’s and Terasaki’s reports. Likewise, induction therapy was
also more prevalent among our patients. Moreover, authors do not give
information about the possible discontinuation of the corticoids therapy in the
post-transplant setting. In our cohort, 70% of patients still had corticoids at 1y
post-transplantation. No data about the incidence, the severity of AR and
recovery of renal graft function were reported in Zou’s and Terasaki’s studies
while they may affect the graft survival (319).
4.4 How to provide evidence of a possible pathogenicity of MICA antibodies
towards the renal graft?
Undoubtedly, to design a study enable to answer to the question whether MICA
antibodies are pathogenic will face the difficulty to clearly isolate the effect of
MICA antibodies on graft outcome as MICA and HLA sensitization are closely
linked. This link has been reflected in all the previous publications related on
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MICA (see annexes). As reported in our second article, preliminary statistical
calculations were performed in order to determine the sample size needed to
detect a difference of death-censored graft survival of at least 5% 3 years after
MICA testing considering a prevalence of MICA antibodies of about 10%, as
showed by Terasaki (291). These analyses revealed that to avoid the possible
confounding impact of anti-HLA antibodies on survival, at least 3500 patients
should have been included to reach ± 170 patients who were HLA-/MICA+.
However, the prevalence of post-transplant MICA antibodies reached about 5%
in our cohort making it should have required 7000 patients to test our hypothesis.
Therefore, we considered such a study unrealistic because it would have
involved huge logistics and financial support.
To design an animal model would also not be devoid of some pitfalls. A major
one would be that mice lack MICA expression. Nevertheless, some might
imagine creating a transgenic mice model since the coding regions for the human
MICA gene are known and could be cloned. Kidneys from transgenic mice could
be transplanted in wild type mice previously immunized with recombinant MICA
antigens and parameters such as albuminuria/creatininuria ratio in daily urine
samples could be screened until sacrifice.
Another mean, but expensive, to study the relationship between MICA antibodies
and graft failure could be the use of a non-human primate model. For example,
monkey donor and recipient pairs should be selected for ABO and HLA
compatibility but MICA mismatching. Recipients should be submitted to
chimerism protocols such as nonlethal total body irradiation, local thymic
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irradiation followed by the infusion of ATG and intravenous donor bone marrow
transplantation before heterotopic renal transplantation and bilateral native
binephrectomy. For example, cyclosprorine administered at day 0 would be
progressively tapered to finally be discontinued. Serum samples would be tested
for anti-donor MICA antibodies, anti-HLA antibodies and creatinine. Renal graft
biopsies would be performed at regular intervals when creatinine is stable and in
addition, when creatinine is rising. We might follow the development of MICA
antibodies in monkeys that remained negative for anti-HLA antibodies and the
appearance of allograft injury.
Just imagine money is not an obstacle and we have huge logistic means such as
a large staff and platforms dedicated to research and highly sophisticated
equipment, we could set up a prospective study through a large collaborative
network that would allow for the recruitment of a huge amount of patients with
available clinical and histological data. Patients should be donor HLA full-
matched and mismatched for MICA with no pre-transplant HLA or MICA
antibodies detected by single antigen flow beads assays. They should receive a
standardized induction and immunosuppressive therapy with clear protocols for
reduction of immunosuppression, especially in cases of infection or cancer.
Patients should undergo routine blood tests every 3 months to assess HLA and
MICA status and renal graft biopsies at Day 0, Month 3, Month 6 and yearly
thereafter, until 10 yr post-transplantation. Only the patients who never
developed HLA antibodies would be kept for analysis. Sera positive for MICA
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antibodies should be controlled for cytotoxicity on cell lines expressing a single
MICA antigen.
To increase the level of evidence for the pathogenicity of MICA antibodies, the
following criteria should be present:
i) Good function over time should be associated with patients who do not develop
MICA antibodies
ii) The appearance of MICA antibodies should precede immune-mediated graft
injury
iii) MICA antibodies should be confirmed as the strongest independent risk factor
for immunological graft loss in an appropriate logistic regression model
iv) MICA+ patients whose graft failed from immune-mediated injury compared
with MICA+ patients who lost their graft from a non-immunological cause or kept
a functioning graft should be confirmed to be DSA
v) MICA+ patients whose graft failed from immune-mediated injury compared
with MICA+ patients who lost their graft from a non-immunological cause or kept
a functioning graft should have high MICA antibodies titers (dose response
effect)
vi) MICA+ patients whose graft failed from immune-mediated injury compared
with MICA+ patients who lost their graft from a non-immunological cause should
have intra-graft MICA antibodies
The ultimate evidence of the pathogenicity of MICA antibodies should be the
demonstration that eliminating de novo antibodies would result in long-term
survival.
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In addition, we may assume that probably MICA antibodies do not act alone but
rather exert their effect in concert with antibodies to other minor antigens
unexplored so far.
Therefore, another way that should be explored to better understand the possible
pathogenic effect of MICA antibodies should be the use of proteogenomics. This
approach combines proteomics and genomics. Proteomics is the systematic
analysis by chromatography and mass spectrometry of all proteins encoded by
the genome in any defined biologic compartment and genomics involves
microarrays, which determines gene expression profile (mRNA transcripts)
correlated to a corresponding level of protein or other available genomic
techniques evolved in recent years such as SNP arrays detecting SNPs of
human genome, alterations in gene copy number or alternative RNA splicing.
The proteomic approach has already been used to screen non-HLA antibody
response in patients undergoing chronic haemodialysis and awaiting a kidney
transplant, to identify compartment-specific non-HLA targets after renal
transplantation and recently, to predict the development of chronic allograft injury
(320, 321).
The proteogenomic approach might be used in order to better understand the
possible role of minor antigens in rejection. A large multi-centre study could be
set up to compare patterns of proteins and antibodies in both serum and graft
between patients with histological features of humoral AR or CR and patients
with normal biopsy. In the event that a specific pattern emerged, it should be
firstly confirmed in a validation sample set and the second step would be to
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identify polymorphisms or aberrations in the genes that encode the selected
proteins or alterations in the translation of RNA transcripts. However, the study
design would require that patients are matched for donor and recipient
demographic characteristics and immunosuppression. Such studies would be
very expensive and time consuming requiring high throughput technology and
advanced bioinformatics analyses for an uncertain expected benefit.
Until now, proteomics has been used in small patient cohort size with most of the
time, the lack of data validation from an independent patient sample set.
Moreover, none of candidate biomarkers identified by proteomics has emerged
so far as a reliable clinical tool for the diagnosis of AR or CR (322-324). This
seems to be related to the inconsistency in sample handling, and the lack of
standardization of experimental design leading to the identification of different
discriminatory proteins by different groups. Current proteomic studies suggest
that a single biomarker does not provide sufficient specificity to distinguish
between diseased and healthy status. Therefore, robust study design with
appropriate statistical power, blinding and validation set is crucial to improve the
reliability of proteogenomic-driven results, making possible the identification of a
panel of candidate markers that will form a signature pattern with sufficient
diagnostic performance and good predictive value.
Although our findings suggest that MICA antibodies do not affect renal graft
outcomes, we cannot exclude that high titer and/or cytotoxic MICA antibodies
might be deleterious in rare patients.
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Therefore, the question is whether it would be reasonable to incur significant
financial and human resources to resolve an issue that concerns finally a low
number of patients for an uncertain expected benefit.
Of interest, it is also unclear how long MICA antibodies persist over time.
Terasaki and colleagues reported that the frequency of post-transplant MICA
antibodies was stable along with time (291). However, nearly two thirds of
patients who were sensitized against MICA before transplantation had no more
MICA antibodies at 1yr post-transplantation in our cohort (data not shown). This
might suggest that MICA antibodies could be absorbed by the graft or MICA
sensitization might be a fluctuating phenomenon. Likewise, in our paper dealing
with the safety of Influenza A/H1N1 vaccine, MICA antibodies were seen to
disappear one month after a positive screening in several kidney transplant
patients and in one haemodialysis patient, advocating for the transitory nature of
these antibodies. We strongly believe that MICA immunisation is highly
influenced by environmental stress and also that immunosuppression modulates
the development of MICA antibodies.
4.5 Influenza A/H1N1 vaccination and MICA sensitization
Our research showed that Influenza A/H1N1-adjuvanted vaccine did not trigger
either an increased production of anti-HLA or MICA antibodies nor induced AR or
a deterioration of renal graft function up to 6 months after vaccination.
Possible MICA sensitization after Influenza A/H1N1 vaccination has also been
recently addressed in cystic fibrosis patients and in lung transplant recipients in
our institution. Analysis of the data confirmed that again transplanted patients
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