Revised diagnostic criteria for

T cell–mediated rejection and

antibody mediated rejection
Dr Mohit Naredi
 Introduction

 The widespread use of potent and specific

immunosuppressive agents has significantly reduced acute
cellular rejection rates and substantially improved 1-year
graft survival following renal transplantation.
 Substantial improvement of long-term (10-year) outcomes,
however, has not been realized
 The ongoing therapeutic challenge is to achieve effective
and safe immunosuppression and avoid unwanted toxicities
to produce enduring renal allograft function
 Introduction

 The incidence of hyperacute rejection caused by preexisting

anti-HLA donor-specific antibodies (DSA) has been nearly
eliminated by crossmatch and compatibility matching
strategies.
 Similarly, the incidence of acute T cell–mediated injury has
been significantly reduced with the effective multimodal
application of immunosuppressive agents.
 However, acute and chronic antibody-mediated rejection
(ABMR) are playing an increasingly critical role in kidney
allograft loss and are considered among the most important
barriers that limit long-term outcomes
Mechanisms of donor-specific antibody-
mediated endothelial injury in renal
allograft
 Donor specific antibodies

 Preexisting or de novo circulating antibodies have been

shown to compromise renal allograft survival.
 These antibodies may be directed against HLA or non-HLA
molecules on endothelial cells, including major
histocompatibility complex class I-related chain A antibody
(MICA), and angiotensin type 1 receptor
 Although DSA are important risk factors for graft loss, the
majority of patients with DSA have stable allograft function
and experience no rejection. It is therefore important to
determine the pathogenic role and specificity of anti-class I
and class II HLA and non-HLA-DSA and to better
understand the effect of de novo DSA compared with
preexisting DSA.
 B cells and plasma cells

 B cells may also contribute to posttransplant immune

regulation through its antigen-presenting function.
 In addition to pathways used by other antigen-presenting
cells (APCs), B cells can present antigen via antigen binding
to the clonotypic B cell receptor.
 In turn, B cells (unlike other APCs) undergo clonal
expansion, which may contribute to rejection by amplifying
antidonor B cell responses
Complement Pathways
 In the classical pathway, immune complexes (Ag-Ab complexes) bind C1 via its C1q
subcomponent, while its C1s protease subunit cleaves C4 and C2. The large C4b
fragment binds to a target and subsequently captures the large fragment of C2
(C2a). This bimolecular complex forms an enzyme (the C3 convertase, C4bC2a) that
cleaves C3 to C3b and releases the anaphylatoxin, C3a. The binding of another C3b
to the convertase (C4bC2aC3b) generates the C5 convertase.
 The lectin pathway is an analogous system, except that the initiating step is the
binding by lectins to repetitive sugars on microbial surfaces. Mannose-associated
serine proteases (MASPs) take the place of the C1 proteases.
 The alternative pathway (AP) continuously self-activates at a low level (a process
called C3 tickover) to generate C3b that deposits on pathogens or debris. C3b
engages the alternative pathway components, factors B (FB) and D (FD), to form a
C3 convertase (C3bBb), which in turn cleaves more C3 to C3b. The binding by
another C3b to the C3 convertase generates the C5 convertase (C3bBbC3b).
Properdin (P) is a positive regulator that stabilizes both the AP C3 and C5 convertases.
The latter subsequently cleaves C5 to release the potent anaphylatoxin C5a, while C5b
engages the terminal pathway and initiates the formation of the lytic membrane attack
complex (MAC).
The complement system is designed to function most efficiently on a biolic membrane.
 Complement pathway

 Once activated, the complement system does not

discriminate between self- and foreign structures, and
it is potentially damaging to both.
 Several molecules exert inhibitory or regulatory effects
on the complement system.
 These substances can be soluble(Liq phase), such as
factor I, factor H, C1INH, CFHR1, and FHL1, or
bound to cell surfaces(solid phase), such as
CD46, CD55, CD59, and complement receptors (CRs)
 They interrupt the formation of C3b by dissociating C3
convertases C3bBb(Alt) and C4b2a(Cl).
 Alternatively, factor I can cleave C3b and C4b to their
inactive forms (iC3b and iC4b).
 C4d:: An Introduction

 C4d is a degradation product of the classic complement

pathway.
 After an Ag-Ab complex fixes complement, a cascade of
events follows, with activation of several complement
proteins.
 Complement protein C4 is split into C4a and C4b. C4b is
then converted to C4d.
 C4d binds covalently to the endothelial and collagen
basement membranes, thereby avoiding removal and
raising the possibility of serving as an immunologic
footprint of complement activation and antibody activity.
 Formation of C4d

 Recleavage of iC4b by factor I generates a soluble

fragment called C4c and another surface-bound
fragment called C4d; the latter covalently binds to
the tissue and can,therefore remain at the site of
complement activation.
 Because the conversion of C4 into C4a and C4b is
event primarily related to the classical pathway (but
also to the lectin pathway), the generation of C4d is
related to antibody mediated complement activation,
for example, in humoral rejection (AMR).
Figure 1
 Covalently bound C4d has a much longer half-life and,
thus, remains at the site of complement activation,
whereas antibodies bind to tissue by hydrostatic, van
der Waals-type of interactions.
 The “footprint effect” of the internal thioester of C4d
becomes strikingly apparent when the blood stream
can clear all soluble/weakly bound molecules quickly,
just like what happens with antibodies on the
endothelial surfaces.
 Covalently bound C4d will not be affected, because it is
anchored tightly to the tissue and, therefore, serves as a
footprint of AMR tissue injury and has been regarded
as “a magic marker” of complement activation and
AMR tissue injury, because of its stability, its strong
association with AMR, and its major impact on graft
survival and patient treatment .
 In normal kidneys, C4d is detectable in the
glomerular mesangium and at the vascular pole.
 This emphasizes that there is constitutive turnover
of immune complexes.
 When the burden of immune complexes increases
with immune complex deposition diseases, C4d
deposition overflows from the mesangium and
vascular pole to the glomerular capillaries (GCs).
 C4d in PTC

 C4d deposition in the glomerular mesangium, the

glomerular basement membrane, the tubular
basement membrane, arterioles, and arterial intima
in both native and transplanted kidneys can be
demonstrated by monoclonal antibody staining and
immunofluorescence of frozen tissue sections .
 Deposition in the PTCs has only been described in
renal allografts and is thought to represent antidonor
humoral activity.(Rem-Graft has high burden of HLA
and non HLA Ag)
 In transplanted kidneys, the reason for the specificity
of C4d staining in the PTC is not entirely clear.
 DSA directly engages HLAs, which are present in the
glomerulus as well as the PTC.
 Anticomplement protection in the PTC is weaker than
in the glomerulus.
 Part of the reason for this is that the glomerulus has at
least four cell-surface complement inhibitors:
1. Decay accelerating factor-CD55,
2. Membrane cofactor protein-CD46,
3. Complement receptor 1 (CR1)- CD35, and
4. Protectin-CD59.
 By comparison, only protectin (CD59) is actively
expressed in the PTC.
 Protectin inhibits the formation of complement
membrane attack complex (C5-9), and, therefore, the
generation of C4d is relatively uninhibited.
 C4d in PTC

 Despite the association between C4d and immune

complex deposition, immunoglobulin has not been
detected in PTC where C4d is detectable.
 The reason for this is unclear and an important
question.
 As humoral rejection probably results from direct
antibody attack on the target endothelial cell,
modulation from the surface may make antibody
detection difficult.
 Endothelial cells dislodge surface antibody
through"capping," "shedding," or "internalization" .
 C4d resists modulation from the surface due to its
covalent binding to tissue structures
DISCOVERY OF C4d AS A CLINICAL MARKER FOR AMR

 Feucht et al(1993) showed that patients with

suspected antibody-mediated injury in the renal
graft had a linear C4d staining pattern in
peritubular capillaries and that the presence of C4d
was associated with impaired graft function.
 Collins et al(2000) tested for presence of C4d
along with other markers of endothelial activation
or injury in renal transplant biopsies suspected of
AMR.
 In 2003 ‘C4d’ was incorporated in the Banff
classification.
 Defining and Diagnosing ABMR-Revised (Banff 2013) classification

Acute/active ABMR; all three features must be present for diagnosis,

1.Histologic evidence of acute tissue injury, including one or more of the following:
 Microvascular inflammation (g > 0 and/or ptc > 0)
 Intimal or transmural arteritis (v > 0)
 Acute thrombotic microangiopathy (TMA), in the absence of any other cause
 Acute tubular injury, in the absence of any other apparent cause
2.Evidence of current/recent antibody interaction with vascular
endothelium, including at least one of the following:
 Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen
sections, or C4d > 0 by IHC on paraffin sections)
 At least moderate microvascular inflammation ([g + ptc] ≥ 2)
 Increased expression of gene transcripts/classifiers, if thoroughly validated
3.Serologic evidence of donor-specific antibodies (HLA or other antigens)
o C4d staining or expression of validated transcripts/classifiers as
o noted above in criterion 2 may substitute for DSA; however thorough DSA testing,
including testing for non-HLA antibodies if HLA antibody
o testing is negative, is strongly advised whenever criteria 1 and 2 are met
 Chronic, active ABMR-

All three features must be present for diagnosis.

1.Morphologic evidence of chronic tissue injury, including one or more of the
following:
 Transplant glomerulopathy (cg > 0), if no evidence of chronic TMA
 Severe peritubular capillary basement membrane multilayering (requires electron
microscopy [EM])
 Arterial intimal fibrosis of new onset, excluding other causes
2.Evidence of current/recent antibody interaction with vascular
endothelium, including at least one of the following:
 Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or
C4d > 0 by IHC on paraffin sections)
 At least moderate microvascular inflammation ([g + ptc] ≥ 2)
 Increased expression of endothelial activation and injury transcripts (ENDATs) or other
gene expression markers of endothelial injury in the biopsy tissue, if thoroughly validated
3.Serologic evidence of donor-specific antibodies (HLA or other antigens)
 Revised Banff 2017 classification of ABMR &
TCMR
Category 1: Normal biopsy or nonspecific changes
Category 2: Antibody-mediated changes
Active ABMR; all 3 criteria must be met for diagnosis
1. Histologic evidence of acute tissue injury, including 1 or more of the following:
 Microvascular inflammation (g > 0 and/or ptc > 0), in the absence of recurrent or de novo
glomerulonephritis, although in the presence of acute TCMR, borderline infiltrate, or infection, ptc ≥ 1
alone is not sufficient and g must be ≥ 1
 Intimal or transmural arteritis (v > 0)
 Acute thrombotic microangiopathy, in the absence of any other cause
 Acute tubular injury, in the absence of any other apparent cause
2. Evidence of current/recent antibody interaction with vascular endothelium, including 1 or more of the
following:
 Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or C4d > 0 by IHC
on paraffin sections)
 At least moderate microvascular inflammation ([g + ptc] ≥2) in the absence of recurrent or de novo
glomerulonephritis, although in the presence of acute TCMR, borderline infiltrate, or infection, ptc ≥ 2
alone is not sufficient and g must be ≥1
 Increased expression of gene transcripts/classifiers in the biopsy tissue strongly associated with ABMR, if
thoroughly validated
3. Serologic evidence of donor- specific antibodies (DSA to HLA or other antigens).
C4d staining or expression of validated transcripts/classifiers as noted above in criterion 2 may substitute for
DSA; however thorough DSA testing, including testing for non-HLA antibodies if HLA antibody testing is
negative, is strongly advised whenever criteria 1 and 2 are met
Chronic active ABMR; all 3 criteria must be met for diagnosis
1. Morphologic evidence of chronic tissue injury, including 1 or more of the following:
 Transplant glomerulopathy (cg >0) if no evidence of chronic TMA or chronic
recurrent/de novo glomerulonephritis; includes changes evident by electron microscopy
(EM) alone (cg1a)
 Severe peritubular capillary basement membrane multilayering (requires EM)
 Arterial intimal fibrosis of new onset, excluding other causes; leukocytes within the
sclerotic intima favor chronic ABMR if there is no prior history of TCMR, but are not
required
2. Identical to criterion 2 for active ABMR, above
3. Identical to criterion 3 for active ABMR, above, including strong recommendation for
DSA testing whenever criteria 1 and 2 are met

C4d Staining without Evidence of Rejection; all 4 features must be present for diagnosis
1. Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or
C4d>0 by IHC on paraffin sections)
2. Criterion 1 for active or chronic, active ABMR not met
3. No molecular evidence for ABMR as in criterion 2 for active and chronic, active ABMR
4. No acute or chronic active TCMR, or borderline changes
Category 3: Borderline changes Suspicious (Borderline) for acute TCMR
 Foci of tubulitis (t > 0) with minor interstitial inflammation (i0 or i1), or moderate-
severe interstitial inflammation (i2 or i3) with mild (t1) tubulitis;
 retaining the i1 threshold for borderline with t > 0 is permitted although this must be
made transparent in reports and publications
 No intimal or transmural arteritis (v = 0)
Category 4: TCMR
Acute TCMR
 Grade IA Interstitial inflammation involving >25% of nonsclerotic cortical parenchyma
(i2 or i3) with moderate tubulitis (t2) involving 1 or more tubules, not including tubules
that are severely atrophic5
 Grade IB Interstitial inflammation involving >25% of nonsclerotic cortical parenchyma
(i2 or i3) with severe tubulitis (t3) involving 1 or more tubules, not including tubules that
are severely atrophic5
 Grade IIA Mild to moderate intimal arteritis (v1), with or without interstitial
inflammation and/or tubulitis
 Grade IIB Severe intimal arteritis (v2), with or without interstitial inflammation and/or
tubulitis
 Grade III Transmural arteritis and/or arterial fibrinoid necrosis of medial smooth muscle
Chronic Active TCMR
 Grade IA Interstitial inflammation involving >25% of the total
cortex (ti score 2 or 3) and >25% of the sclerotic cortical
parenchyma (i-IFTA score 2 or 3) with moderate tubulitis (t2)
involving 1 or more tubules, not including severely atrophic
tubules; other known causes of i-IFTA should be ruled out
 Grade IB Interstitial inflammation involving >25% of the total
cortex (ti score 2 or 3) and >25% of the sclerotic cortical
parenchyma (i-IFTA score 2 or 3) with severe tubulitis (t3)
involving 1 or more tubules, not including severely atrophic
tubules; other known causes of i-IFTA should be ruled out
 Grade II Chronic allograft arteriopathy (arterial intimal fibrosis
with mononuclear cell inflammation in fibrosis and formation of
neointima)
Acute and
chronic
definitions
of ABMR
based on
C4d
positivity
 C4d staining without evidence of rejection

All three features must be present for diagnosis

1.Linear C4d staining in peritubular capillaries (C4d2 or
C4d3 by IF on frozen sections, or C4d > 0 by IHC on
paraffin sections)
2.g = 0, ptc = 0, cg = 0 (by light microscopy (LM) and by
EM if available), v = 0; no TMA, no peritubular capillary
basement membrane multilayering, no acute tubular
injury (in the absence of another apparent cause for this)
3.No acute cell-mediated rejection or borderline changes
 Revised Banff Criteria for ABMR in Renal
Allografts: Inclusion of C4d-Negative ABMR

 C4d-negative ABMR is incorporated into the Banff

classification for renal ABMR.
 Immunohistopathologic evidence (typically in the form
of C4d staining), previously required for dx of ABMR,
has been replaced by a category of current/recent evidence
of antibody interaction with the endothelium, the latter
including C4d positivity but also at least moderate MVI or
elevated expression in the biopsy tissue of gene transcripts
indicative of endothelial injury.
 C4d is scored semiquantitatively in four categories

 No C4d staining (0% of peritubular capillaries)

 Minimal C4d staining (0–10% of peritubular
capillaries)
 Focal C4d staining (10–50% of peritubular
capillaries)
 Diffuse C4d staining (>50% of peritubular
capillaries)
C4d scoring in PTC and influence of staining method.
 Controversy

 Although a diffuse C4d staining is defined as

positive, the definition and clinical significance of
‘minimal’ and ‘focal’ C4d staining remain debated
issues.
 Most experts consider a focal staining pattern as a
red flag, especially when detected on paraffin-
embedded tissue or in the presence of donor-specific
antibody and/or suspicious histopathological
features.
 Immunohistologic findings in acute AMR. (A) C4d staining in PTCs.
Staining is strong, linear, and diffuse (indirect IF) (B) IgG deposition in the
wall of a small artery showing early fibrinoid necrosis
 Timing of deposition

In sensitized individuals C4d deposition can be

evident within one hour of transplantation.
 ABMR Classification and Phenotypes

 In previous Banff report, two principal phenotypes of

acute ABMR were defined-
 (1) ABMR phenotype 1- in the presensitized patient,
occurring early posttransplant; and
 (2) ABMR phenotype 2- which develops from the
emergence of de novo DSA in the late posttransplant
period and is thought to be mostly related to
nonadherence or inadequate immunosuppression.
The natural history of phenotype 2 ABMR.
 Other organ Tx

It is recommended that every renal, cardiac, and

pancreas allograft biopsy should be stained for C4d.
C4d staining is considered positive only
when depositions are found in the following
anatomical locations:
Kidney: Peritubular capillaries .
Pancreas: Interacinar capillaries .
Heart: Myocardial capillaries.
 Focal staining

A-In paraffin-embedded tissue combined with a

positive test for DSA and histopathological features:
Most laboratories consider this as ‘positive for AMR.
It is advised to treat the patient.
B-In frozen tissue combined with a positive test for
DSA and
histopathological features: This is sometimes called
a ‘probable AMR’. Most experts would consider
treatment, however, prospective
studies should investigate this group further.
 C4d positivity without detectable DSA

(a) Possible explanations:

(i) Allo-antibodies are present, but they are not detected by
standard anti-HLA assays (for example, anti-endothelial
antibodies).
(ii)Allo-antibodies are absorbed by the graft, as is
sometimes shown by reappearance of allo-antibodies
in the blood after graft nephrectomy.
(iii) Allo-antibodies are not present and C4d deposition is
caused by something else than DSA (for instance
autoimmune disease,i.e Lupus nephritis or any form of
lectin pathway activation).
 b.How to proceed?

(i) In case of diffuse C4d staining in PTC and

histological evidence for AMR: Most experts would
advise to treat the patient for AMR.
(ii) In case of focal C4d staining and histological
evidence for AMR: Check for other possible
underlying diseases. If no other cause can be found:
Treat the patient for AMR.
(iii) In case of focal or diffuse C4d staining and
absence of histological changes the decision as to
whether to treat for AMR is more uncertain. Treating
or close follow-up are both suitable options.
 C4d positivity in grafts without histological abnormalities

(a) In ABO-incompatible grafts: In case of no histopathology and no

graft dysfunction: It is suggested to interpret this as ‘graft
accommodation’. No treatment is necessary, but close follow-up is
strongly advised, as it is unknown what happens with these grafts
during long-term follow-up.

(b) In positive cross-matched patients (presensitized patient), this

should not be seen as graft accommodation and is a rare and more
worrying situation than the above: In case of normal histology
and no graft dysfunction: Interpret as probable AMR and consider
treating the patient. Prospective studies on the long-term follow-up
of this group of patients are awaited
C4d staining patterns in different clinical settings
Use of Molecular diagnostics
 Novel non invasive markers

 Functional cell-based immune monitoringinterferon (IFN)-gamma enzyme-

linked immunospot assay (ELISPOT), which measures IFN-gamma secretion by
recipient T cells in response to donor antigens
 Kidney Solid Organ Response Test (kSORT)
 Donor-derived cell-free DNA
 uantification of microRNAs (miRNAs) in peripheral blood
 global gene expression profiling
 Urine messenger RNAs
 Urine microRNAs
 Urine proteins — A number of urinary immune-related proteins have been
identified as biomarkers of acute rejection of the renal allograft [64]. As examples,
elevated urinary levels of chemokine (C-X-C motif) ligands 9 and 10 (CXCL9 and
CXCL10) 8 proteins (PIGR, CP, GPRC5A, APOA1, TSPAN1, TTR, AZGP1 and HPX)
 Urine proteomics/peptidomic including fragments of collagens, beta-2-
microglobulin, alpha-1-antichymotrypsin, and uromodulin.
 Imaging method
 18-fluoro-2-deoxyglucose (FDG) positron emission tomography
 31-phosphorus magnetic resonance spectroscopy (MRS)
 Predictors of poor outcome related to
antibody-mediated injury

 Acute and chronic ABMR are associated with poor

outcomes after kidney transplantation. Specifically,
patients with acute ABMR are at greater risk for subsequent
rejection, chronic ABMR and graft loss
 Those with chronic ABMR are at increased risk for graft
loss
 Predictors of poor outcome related to
antibody-mediated injury

 C4d and microcirculation inflammation are independent

biomarkers of subsequent rejection, chronic ABMR and
graft loss in patients with acute ABMR
 C4d and endothelial injury are also associated with poor
outcomes in chronic ABMR
 Strategies to prevent ABMR

1. Do not transplant highly sensitized patients

2. Avoid blood transfusion
3. Paired kidney exchange
4. In sensitized patients, precise characterization of their
alloantibodies and exact HLA typing of the donor at the
time of transplantation
5. Participation in special programs (such as the
Eurotransplant Acceptable Mismatch Program)
6. Removal of DSA (plasmapheresis, immunoadsorption)
 Strategies to prevent ABMR

7. Direct or indirect inhibition of DSA production a. Anti-B

cell agents (rituximab ) b. Anti-plasma cell agents
(proteasome inhibitors, e.g. bortezomib) c. Rabbit anti-
human thymocyte immunoglobulins (e.g. thymoglobulin)?
d. Costimulation blockade (e.g. belatacept)?
8. Inhibition of complement cascade (eculizumab )
9. Intravenous immunoglobulin e. Neutralizing DSA: anti-
idiotypic activity f. Inhibiting complement activation by
binding C3b, C4b g. Inhibiting activation of macrophages,
neutrophils by binding FcgRs h. Apoptosis of B cells
(inhibits CD19 expression)
10. Splenectomy
 Treatment of Chronic ABMR

 Chronic ABMR is a more difficult condition to treat because

irreversible tissue damage has occurred in the setting of
severely compromised graft survival.
 A small-scale retrospective study of rituximab combined
with standard maintenance immunosuppression (including
prednisone, mycophenolate mofetil and calcineurin
inhibitors) in 31 patients with chronic ABMR had
encouraging results, with partial therapeutic response and
an increase in median graft survival in the rituximab-
treated group compared with the control group (685 days
vs. 439 days, respectively).
 Treatment of Chronic ABMR

 The outcomes within the rituximab group were

dichotomous, with significantly different median survival
time in responders compared with non responders and
control patients, though there were no pathologic
parameters that distinguished any subset of patients
 Clinical trials of rituximab for the treatment of chronic
ABMR are ongoing
 Summary and take home points

 C4d is a widely used marker for antibody-mediated rejection in the

kidney, heart, pancreas, and possibly lung allografts.
 In ABO-incompatible grafts, C4d is not a useful test, and may even
indicate graft accommodation.
 In pregnancy, C4d at the fetal–maternal interface indicates
antibody-mediated rejection of ‘the fetal allograft’, as was
demonstrated in antiphospholipid antibody-induced fetal loss.
 C4d shows that the complement system is involved.
 If complement targeted therapies will be part of our future
treatment options, a marker such as C4d will be needed to identify
patients susceptible for those kind of (expensive) treatments.
 Alternatives for C4d are emerging (genomics, molecular diagnostics,
and endothelial transcripts) and if proven useful, effort will be made
to transform these techniques or their progeny to practical tests.
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