MICA Antibodies in Renal Transplantation

The role of MIC-A in renal allograft loss -
More than meets the eye?

Christos Argyropoulos

Renal Grand Rounds 12/1/08

11/30/08 C:UserschrisargDocumentsMIC-A grandrounds.ppt page 1

The Case of Mr RC I
Patient is a 45 y/o wm diagnosed with LCD in April 2000.
a) Bone marrow biopsy (2000): increase in the number of bone
marrow plasma cells (7%), with normal cytogenetics.
b) Native kidney biopsy : the glomeruli were hyperlobular and had
eosinophilic material ( Congo red and Thio-T stains were negative)
and wrinkling of the basement membrane . There was marked
mesangial cellularity, with mesangial and tubular material deposition.
Immunofluorescence staining of glomerular and tubular membranes
was positive for kappa light chains and C3 was negative (C4 was not
done).
At that time he received 4 doses of VAD chemotherapy which led to AKI
from which the patient never recovered and was started on Hemodialysis
in 8/00.
Patient transplanted in 4/10/6 and was maintained on FK/MMF since
then.
Graft characteristics (DD, age 43, CIT1641, mismatched for A1,B1,DR2)
Patient’s baseline Scr between 1.5 – 2.1 up to 8/07


The Case of Mr RC II
From 9/07 until 8/05/08 patient’s Scr increased gradually
up to 3.3
Rapid deterioration in renal function noted at the
beginning of August: 3.8(8/7/08)→5.1 (8/11/08).
ELISA Class I,II screens : 0/0
Luminex Class I,II : (-)/weakly (+)
Luminex MICA : weakly (+)
Cylex: 371
Urine Protein/Creatinine ratio : 66/87.7


The case of Mr RC III
Allograft Biopsies (x2) :
GLOMERULOPATHY WITH INCREASED MESANGIAL
CELLULARITY AND INCREASED MESANGIAL MATRIX
NO EVIDENCE OF ACUTE REJECTION (i0 t0 v0 g0).
MILD CHRONIC ALLOGRAFT NEPHROPATHY (ci1 ct1 cv1).
MILD ISOMETRIC TUBULAR VACUOLIZATION WITH
SCATTERED PROTEIN RESORPTION DROPLETS
C4D stain was negative.
Borderline ACR noted in the first biopsy


On the basis of this biopsy and the positive MICA screen patient was
treated as a possible MICA releated rejection with SM pulses.

Patient is pulsed with steroids on 8/0808 with reduction in serum
creatinine to 3.5 (8/15/08).


MAJOR HISTOCOMPATIBILITY COMPLEX
CLASS I CHAIN-RELATED GENE A: MICA

Discovered in 1994 by a GenBANK search for
sequences related to the MHC class I genes.
Named MIC (MHC class I chain related genes) due to
their chromosomal location (6p21.3 in a 200kb region
spanning the TNFA/B cluster, close to the HLA-B
locus)
MICA encodes a 383-aa cell membrane polypeptide
with a molecular weight of ~ 43 kDa.
MICA is highly polymorphic in its extracellular domain
and at strong linkage disequilibrium with HLA
haplotypes in specific populations.
MICA does not interact with the T Cell Receptor (TCR)
but with NKG2D (C-type lectin activatory receptor)
found in NK cells, Tγδ and CD8+ Tαβ cells.


Transcription Pattern of MIC-A in Normal
Human Tissues and Tumours

PLoS ONE. 2007; 2(6): e518.
doi: 10.1371/journal.pone.0000518.


Effector functions of NKG2D
engagement by NKG2DL (MICA)
Engagement of NKG2D by
MICA leads to enhanced
cytolytic activity of NK and
Tγδ cells
This activity is directed
against virally infected (e.g
CMV) and tumour cells in
multiple experimental
systems. 
Engagement of NKG2D is transduced
Exposure to carcinogens by DAP10 (a S-S bonded homodimer)
upregulates MICA that features a YXXM motif that interacts
expression, while NKG2D- with P85 PI3K.

In CD8(+) T cells, NKG2D may plays
deficient mice develop the same role as the the co-stimulatory
tumours molecule CD28.

NKG2D (but not CD28) engagement
Cell mediated killing through
induces Immunological Synapse
NKG2D is inhibited in the formation in T cells.
presence of HLA-I 
NKG2D signaling is enhanced by IL-15
molecules (“missing-self”). or high doses of IL-2 converting T cells
into NK like cytotoxic effector cells


Signal Transduction cascade of NKG2D
signaling in NK cells

Trends in Molecular Medicine Vol.14 No.4 179-189


NKG2D ligands and their regulation

Trends in Molecular Medicine Vol.14 No.4 179-189


Non-HLA Antibodies In Renal
Transplantation
Mismatch against non-HLA antigens can lead to
rapid graft loss in experimental models of skin, and
cardiac transplantation.
In clinical renal transplantation >10% of allograft
biopsies with C4d deposition will not be associated
with anti-HLA antigens.
It is estimated that 38% of chronic graft loss is due
to antibody formation against non-HLA targets.
Candidate clinically relevant non-HLA antigens are
present in non-lymphocyte cellular populations (e.g.
the Endothelial – Monocyte (EM) antigen system.
Routine cross-matches are performed against T-
lymphocytes not endothelial cells, hence non-HLA
mismatch will go unrecognized.


Anti-MICA as non-HLA Abs
MICA molecules are expressed in endothelial cells
which is the target of the recipient's immune
response in acute (and chronic) humoral rejection
The MICA system is highly polymorphic and thus
there is a high probability that a donor and the
recipient will be mismatched for these antigens.
If the MICA system is implicated in graft loss, the
following conditions should be met:
a. Antibody formation should precede organ dysfunction
b. Antibody formation should predict organ dysfunction
c. Antibody and/or antigen/antibody complex should be
eluted from the target organ during episodes of acute
dysfunction
d. There should be an approximate dose - response curve
between antibody level and progression of organ
dysfunction

Immunologically mediated tissue injury
vs rejection
Conditions 1-4 are necessary conditions for an
immunologic mechanism of tissue tissue injury.
These conditions are met by clinically important
antibodies in acute and chronic antibody mediated
diseases : ITP, AIHA, serum sickness, SLE, anti-
GBM disease, post-infectious GN, Reiter's
syndrome, Heymann nephritis, Goodpasture
syndrome, immune hydrops fetalis, hyper-acute
allograft rejection.
Autologous (anti-GBM disease), heterologous (post-
infectious GN) or allogenous (anti-HLA) antibodies
may lead to immunologically mediated tissue injury.
For an antibody to qualify as responsible for
allograft rejection, its specificity against the donor
(Donor Specific Antibody - DSA), rather than the
recipient should be established.

Consequences of non HLA AB binding to
the graft
a. Antibody formation
b. EC activation
c. Expression of CAM
d. Expression of
cytokines
e. Activation of
coagulation
cascade
f. Migration of
inflammatory cells
to the graft
g. Tissue destruction
via complement
activation,
thrombosis,
Current Opinion in Immunology 2008, 20:607–613
cytolysis (relevant to
MICA)

First clinical Studies of MICA Ab testing
in renal transplantation I
First study (1) that examined the positivity of MICA
in allograft recipients published in 2000


In 3/56 renal transplant recipients the specificity of
the MICA antibody was determined : they harbored
a MICA alloantibody i.e. a DSA.
Caveat : cross-reactivity and specificity of MICA-
Abs not rigorously assessed. In 2/3 patients the
investigators did not test the patient's own MICA for
cross - reactivity
(1) Human Immunology 61, 917–924 (2000)


in renal transplantation II
In one patient multiple serum specimens were
available allowing a longitudinal assessment.

Rejection



in renal transplantation III
Retrospective analysis of 748
serum samples from 139 pts
(115 first grafts) assessed at
various points after transplant.
69 (49.6%) of patients had one
or more ACR (within the first 3
mos)
Detection of MICA antibodies:
flow, blocking, WB
8/13 of grafts lost to
Ability of MICA-Ab to induce a “irreversible rejections”
tested positive, compared
prothrombotic phenotype on to 1/7 grafts lost to death
renal endothelial cells was with a functioning graft
assessed Pretransplant titers were
much lower (1:50)
compared to postrejection
titers (>1:2000)
(1) Transplantation Vol. 74, 268–277, No. 2, July 27, 2002


in renal transplantation IV

In 5 cases of irreversible graft loss the anti-endothelial
AB was directed against MICA.


in renal transplantation V
Functional significance of anti-MICA antibodies:
1. Complement cascade
2. Induction of a prothrombotic phenotype in kidney endothelial cells
(reduction of antithrombotic and induction of prothrombotic surface
molecules)



Detection of anti-MICA relative to the
time of transplant I
Time of detection relative to transplantation
important to differentiate between
auto/heterologous sensitization (antibody present
before transplant), and allosensitization (likely when
Ab detected after surgery).
Autologous antibodies present before transplant are
not DSAs technically (although they may bind to
donor antigens).
The clinical scenario of the patient with non-MICA
autoantibodies detected before transplant is more
likely to be similar to anti-GBM disease post
transplant than HLA allosensitization.
On the other hand MICA antibodies detected after
surgery could either be bona fide pathogenetic
DSAs or immune markers of tissue injury.


time of transplant II
Question has been addressed in various studies to
data. In (1), sera from patients on the waiting lists,
pts with functioning grafts and allograft
nephrectomies were examined retrospectively:

In 1 case assessed there was indirect evidence that anti-MICA was a DSA


time of transplant III
Anti-MICA antibodies are also detected after
transplant.
In a prospective study of 1319 patients assessed for
anti-HLA and anti-MICA antibodies anti-MICA was
detected in ~9% and was constant over time

(1) American Journal of Transplantation 2007; 7: 408–415


time of rejection I
The temporal evolution
of anti-MICA positivity
was examined retro-
spectively in patients
with negative pre-txp
anti-HLA and graft
survival> 1000 days
39 pts who rejected
were matched against
26 simultaneous control
patients with well
functioning grafts.

American Journal of Transplantation 2005; 5: 2265–2272


time of rejection II
Antibody could increase or decrease with time and could
even disappear.
HLA (+) : 72 % rejection versus 46% (controls)
HLA/MICA (+) : 77% (rejection) versus 46% (controls)

MICA noninc MICA inc
MICA (-) MICA (+) Rejected 8 3
Rejected 2 9 Did-not reject 14 0
Did-not reject 11 3

P-value = 0.005 P-value = 0.07
American Journal of Transplantation 2005; 5: 2265–2272


time of rejection III
Single center retrospective assessment of anti-MICA(+)
in 173 patients who received a K or PK (both first
transplants and re-transplants )from 1/98-3/03.
Patients classified according to functional status and
timing of serum collection :
“Functioning” (n=82) : stable allograft function
“Before – failure” (n=62) : dead or patients with ≥1 rejections
“After – failure” (n=28) : specimen available after patient had
returned to dialysis
Biopsies and C4D staining were not uniformly available;
authors assumed that all grafts were lost to “chronic
rejection”
ISP was the standard protocol of CSA + MMF/AZA +
Steroids + Basiliximab induction
Human Immunology 67, 683–691 (2006)


time of rejection IV

Anti-HLA was detected with FlowPRA or Luminex.

All other Abs were detected by cell dependent cytotoxicity



Summary of early studies regarding the
detection of anti-MICA
MICA antibodies may develop before or after transplantation at a low
rate (5-20%) and titers may increase after rejection.
Poor graft survival may be associated with positive MICA screens
MICA antibodies do not demonstrate a consistent dose response
relationship between graft survival/function and antibody level in
either individual patients or at the population level.
On the other hand, there appears to be a dose response curve in
anti-MICA titers in anti-HLA(+) patients who lose their grafts.
There is only one ex vivo study looking at complement fixation by
anti-MICA isolated from patients (N=5).
Complement fixation by anti-MICA has been shown in heterologous
systems(1). In these studies mice were immunized with human MICA
antigens followed by assessment of cellular proliferation, cytotoxicity
and complement activation. All these functions were enhanced in a
MHC-I restricted manner. Note that the MICA locus is absent from the
murine genome and the orthologous gene H-2 is 33% identical to
human MICA. Is this experiment relevant to clinical scenarios?


Clinical Significance of positive MICA
antibody screens pre – transplant I
Retrospective study of 1910 diseased donor kidney transplant
recipients recipients who underwent transplantation between
1990 in 20 centers in 13 countries.
Anti-HLA and anti-MICA were determined by ELISA and
Luminex assays respectively
Allograft function was assessed by serum creatinine levels at
3,6,12 months. Biopsy findings were not reported
23 pts (1.2%) and 34 (1.8%) were lost to f/u respectively
Patients with anti-MICA antibodies had a 1-year graft-survival
rate of 88.3±2.2% as compared with 93.0±0.6% among
patients in the MICA antibody–negative group (P = 0.01).
When only recipients of first transplants were considered, the
graft survival rate was 87.8±2.4% among 183 MICA
antibody–positive patients as compared with 93.5±0.6%
among the 1473 recipients who were MICA antibody–negative
(P = 0.005).

N Engl J Med 2007;357:1293-300.


antibody screens pre – transplant II

N Engl J Med 2007;357:1293-300.


antibody screens pre – transplant III

In summary MICA (+) before transplant was associated with poor graft function.
The risk appeared to be higher in patients well matched for the HLA
N Engl J Med 2007;357:1293-300.


Functional Significance of anti-MICA(+)
detected after transplantation I
Similar to the case of anti-MICA(+) detected in the
pretransplant period, anti-MICA(+) after transplantation
is associated with poor outcomes.
Most of the data come from two prospective multicenter
study of 1329 and 2389 patients with functioning
allografts enrolled in the 13th and 14th International
Histocompatibility Workshop (1) . Selection criteria: (-)
anti-HLA preTxp and normal renal function 6 months
post TxP.
The data from this cohort were analyzing under the
assumption that anti-MICA positivity has the same
significance as a positive anti-HLA test.
In particular, renal survival of patients with (-) anti-MICA
was not compared against patients with (+) anti-MICA
but against renal survival of pts with either anti-HLA or
anti-MICA positivity.


detected after transplantation II

Effects of MICA (+) limited to DD recipients


detected after transplantation III

Effects of MICA (+) limited to DD recipients


detected after transplantation IV

The only reasonable conclusions that can be drawn from this study are : anti-HLA
(+) is associated with poor allograft survival, and that anti-MICA (-) is associated
with better survival that anti-HLA (+) (1) American Journal of Transplantation 2007; 7: 408–415


detected after transplantation V
185 recipients of LR allograft with negative pre-transplant
cross match and survival at 6 months post TxP
ISP: Combination of 3 drug regimens, no induction. Rejections
Tx with SM pulse x 3 days, and refractory cases with ATG or
OCT3
Bx was performed in all patients with unexplained rising Scr.
Banff grade of rejection: I(6), 19(IIA), 16(IIB), 1 (III)



detected after transplantation V


Overall 16% were anti-MICA (+).

40% of anti-MICA(+) patients lost their graft compared to 14% of anti-MICA (-)
patients



Relationship between positivity for anti-
HLA Abs and anti-MICA I
In (1) looking at pre-transplant MICA sensitivity:
1.9% patients were (+) anti-class I and anti-MICA, and 1.8%
harbored both (+) anti-class II and anti-MICA
16.8% (37/220) of patients with anti-class I were MICA(+), versus
10.7% (180/1684) with no anti-class I (p=0.007)
17.2% (35/204) of patients with anti-class II were MICA(+) versus
10.8% (182/1692) of those without anti-class II (p=0.007)
Other studies also showed (2) such an association when anti-MICA
specifities were analyzed while others did not examine the issue (3)
In the study (3) with the longest follow-up (10 year), OR(anti-MICA (+)|
anti-HLA(+)) was 0.17 (p-value 0.04) in patients who rejected, but 1.79
(p-value=0.66) in patients with functional allografts.
In another study (4) of predominantly anti-HLA (+) (93% out of 266)
patients, anti-MICA was detected in 21% with graft loss and 7% without
graft loss. All pts with anti-MICA were (+) for anti-HLA

(1) N Engl J Med 2007;357:1293-300. (3) American Journal of Transplantation 2005; 5: 2265–2272
(2) Human Immunology 68, 362–367 (2007) (4) Clin Transpl. 2006:265-90


Relationship between positivity for anti-
HLA Abs and anti-MICA II


There seemed to an interaction between anti-MICA positivity and anti-HLA
positivity in that the % of rejection and graft loss was higher when patients were
doubly positive.



Mechanisms responsible for anti-MICA
antibody generation I

The following explanations for the development of anti-
MICA have been entertained in the literature:
Allosensitization from transfusions (similar to anti-HLA)
Allosensitization from pregnancies
Allosensitization from Donor Antigens (this is the main
hypothesis entertained in the transplant literature)
Development as a marker of immune-mediated cell death
(e.g. by anti-HLA)
Development as a regulatory mechanism for the innate
arm of immunity (NK Tγδ and CD8+ Tαβ cells).
Development as a result of auto-immunity.


antibody generation I
Anti-MICA as a result of blood transfusion is the least
likely explanation (the antigen is not present RBCs, is
not expressed at the protein level in lymphocytes or
monocytes (or so we think).
Explicitly tested in the pre-transplant series published in
N Engl J Med 2007;357:1293-300.

Transfusions were associated
with anti-HLA but not anti-
MICA antibody formation


antibody generation II
Prior pregnancies (and multiparity) are plausible explanations for
the development of this antibody before transplant in women.
In the pre-transplant series published in N Engl J Med
2007;357:1293-300.there was a slightly higher incidence
of anti-MICA positivity in men rather than women
The level of anti-MICA in pregnancy and anti-HLA (-)
patients was prospectively examined . Only one
specificity could be deemed as significantly different
from the controls:


The case of Mr RC (cont'd)
Serum Protein Electrophoresis : decreased
globulins, no monoclonal spike
Serum free kappa light chain: 134 (normal < 19.4)
Serum free lambda light chain: 11.4 (normal < 26.3)
UPEP : Possible low concentration of monoclonal
protein (but negative immunofixation), and
glomerular proteinuria
Urine b2-microglobulin: 2.66 (normal < 0.23)
Serum b2 microglobulin: 10.1 (normal < 2.5)
Severely depressed serum immunoglobulin levels


The case of Mr RC (cont'd)
Bone Marrow Biopsy (9/2/08):
INVOLVED BY A KAPPA LIGHT CHAIN RESTRICTED
PLASMA CELL NEOPLASM.
NORMOCELLULAR BONE MARROW WITH TRILINEAGE
HEMATOPOIESIS.
MARKEDLY REDUCED IRON STORES.
11.3% Plasma Cells

Patient's final diagnosis was recurrent renal disease
due to PCD (multiple myeloma). He was started on
Dex/Velcade by Hematology, and has been on
Hemodialysis since 10/10/08
Is the MICA antibody causally related to the patient’s
graft failure or a coincidence ? Can anti-MICA develop
in non transplant settings, and if so what's is function ?


Anti-MICA formation in tumor immuno-
surveillance and immunity I
In vitro anti-MICA coating of
tumor cells (1) enhances DC
tumor antigen uptake and
presentation (“DC-vaccine
adjuvant”)
Tumour specific CD8 cells are
generated at increased
frequency after DC priming with
anti-MICA
These CD8 cells demonstrated
increased tumor cytolytic
activity, IFN-gamma production
Tumour specific CD4 responses
were also observed

(1) PNAS May 3, 2005 vol. 102 no. 18 6461–6466
(2) PNAS June 13, 2006 vol. 103 no. 24 9190–9195


surveillance and immunity II
In (2) a patient participating in
an experimental protocol for
metastatic melanoma with
CTL4-Ig (costimulation
blockade) was shown to
improve after the development
of antibodies against the MICA
antigen.
Clinical improvement was
associated with decreases in
soluble MICA antigens and
increases in anti-MICA
antibody.
Surface expression of NKG2D
receptor increased after CLTA4-
Ig administration
(1) PNAS May 3, 2005 vol. 102 no. 18 6461–6466
(2) PNAS June 13, 2006 vol. 103 no. 24 9190–9195


surveillance and immunity III
NK activity was
restored to normal
in the presence of
anti-MICA

In summary anti-MICA
production by
costimulation blockade
IFN-gamma enhances NKG2D
production by mediated immunity by
CD8(+) T cells decreasing the levels
against tumor of the (immuno-
antigens was suppressive) tumour
restored derived soluble MICA
Ag.
(1) PNAS May 3, 2005 vol. 102 no. 18 6461–6466
(2) PNAS June 13, 2006 vol. 103 no. 24 9190–9195


The role of sMICA/anti-MICA in PCD I
The role of sMICA/anti-MICA in the progression of
PCDs (MGUS to myeloma) was recently examined
(1)
Normal plasma cells do not express MICA, where
as MGUS plasma cells express surface MICA and
myeloma cells expressed cytoplasmic MICA

Soluble MICA was not detected in pts with MGUS
but was detected in patients with myeloma.
Soluble MICA antigen positivity was associated with
depressed NKG2D dependent immune responses

(1) PNAS 2008 Jan 29;105(4):1285-90


The role of sMICA/anti-MICA in PCD II

Opsonization of
MICA (+) cells

(1) PNAS 2008 Jan 29;105(4):1285-90


Anti-MICAs and immunologically
mediated native kidney disease I
Patients with Wegener's granulomatosis and other forms
of vasculitis demonstrate high numbers of MICA(+)
circulating endothelial cells during disease activity (n=16)
but not disease remission (n= 36)
These cells produced higher levels of IL-8, ENA-1, MIP-1,
and GRO- in active disease compared to remission and
healthy controls.
The numbers of VAP-1/MICA
cells were inversely correlated
with hemoglobin levels
(ρ=0.51, P<0.001) and
positively with leukocyte
count (ρ=0.5, P = 0.015) and
CRP (0.41, P=0.01).
Furthermore, VAP-1/MICA cells
were significantly and
positively correlated with the
number of organs involved
(ρ=0.53, P <0.001)
J Am Soc Nephrol 16: 3110-3120, 2005


Anti-MICAs and immunologically
mediated native kidney disease II
During active disease,
MICA is upregulated in
peritubular endothelium
and glomerular capillary
cells
Patients with active
Wegener's will also have
a high % of circulating
anti-endothelial
antibodies (of unknown
molecular specificity)
To date the % anti-MICA
positivity of patients with
Wegener's and vasculitis
has not been determined
J Am Soc Nephrol 16: 3110-3120, 2005
J Am Soc Nephrol.18(9):2497-508,2007


Conclusions I
MICA antibody positivity is a novel marker associated
with graft loss after renal transplant in ~20% of renal
transplant patients
Anti-MICA (+) confers poor prognosis irrespective of the
time they are detected (before or after transplant)
The source of Anti-MICA(+) pre-Txp remains elusive but
interesting possibilities include:
Innate immune system effector functions relating to
immunosurveillance of solid tumors and plasma cell disorders
Pathogenetic (auto-antibodies) against renal tissue (as in
Wegener's)
A marker of immune cell activation against cell death


Conclusions II

The source of Anti-MICA(+) appearing de-novo post-Txp also
remains elusive. It could represent :
A bona fide DSA that requires special attention to immune monitoring
after transplant (routine Luminex assays)
A marker of immune cell activation e.g. due to anti- HLA mediated
allograft damage
Management of the anti-MICA positive patient in either the
pre-op or the post-op setting is far from clear.
Further studies will be needed to examine the relation between, type of
native renal disease, occult malignancy and appearance of anti-MICA
before transplant.
In the post-op setting, it is not clear whether intensification of ISP is
required and which adjunctive therapies should be used. If anti-MICAs
are causally implicated in allograft loss , then recombinant sMICA could
be a specific therapy
Given preliminary data from native kidney disease, allograft Bx staining
for MICA should be examined in a prospective fashion.


MICA Antibodies in Renal Transplantation

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