REVIEWS

B cells and transplantation tolerance

Allan D. Kirk, Nicole A. Turgeon and Neal N. Iwakoshi
Abstract | Transplantation tolerance is a state of immune unresponsiveness (or benign responsiveness) to the
presence of specific, nonself antigens in the absence of chronic immunosuppressive therapy. Renal transplant
tolerance remains a desired yet generally unattained goal that would enable transplantation to be performed
without the risk of graft rejection or the need for broadly immunosuppressive drugs, which can have toxic
effects. Studies published in the past few years have provided evidence that B cells have an important role
in both graft rejection and transplantation tolerance. Indeed, antibody-dependent and antibody-independent
functions of B cells account for both tolerogenic and rejection-promoting immune responses in transplant
recipients. This Review comprises a discussion of the mechanisms involved in the induction of B-cell tolerance
and a survey of current and emerging therapies that target the effects of B cells in transplant recipients.
Kirk, A. D. et al. Nat. Rev. Nephrol. 6, 584–593 (2010); published online 24 August 2010; doi:10.1038/nrneph.2010.111

Introduction Physiological self tolerance

In the past, T‑cell‑mediated mechanisms were con‑
sidered the main cause of renal allograft rejection.
Although improvements in T‑cell‑directed immuno‑
suppression have decreased the incidence of acute
cellular rejection,1,2 humoral immune responses, specifi‑
cally those mediated by alloantibodies (that is, by anti‑
bodies produced by the graft recipients that react with
graft isoantigens), have increasingly been recognized
as causes of renal allograft rejection. Indeed, humoral
immune responses can lead to renal allograft rejection
even in patients whose cell‑mediated immune responses
are well controlled.
The overall incidence of antibody‑mediated rejec‑
tion (AMR) is estimated to be 2–10% for kidney trans‑
plant recipients.3,4 The frequency of AMR is increased
in individuals with a previously failed allograft and in
sensi tized patients (that is, those who have allo‑
antibodies before renal transplantation as a result of
pregnancy, blood transfusions or prior transplantation
procedures). AMR presents as graft dysfunction in the
presence of donor‑specific antibodies, and its presence
is indicated in biopsy samples by evidence of comple‑
ment deposition (typically deposition of C4d).5–7 The
development of donor‑specific antibodies and histo‑
pathology consistent with AMR correlates with poor
long‑term allograft survival. In this Review, we provide
an overview of the physiological mechanisms of self tol‑
Emory Transplant erance and describe the currently available and emerg‑
Center, Department of
Surgery, Emory ing therapies that target B cells in the setting of renal
University, transplantation. A complete discussion of the mecha‑
101 Woodruff Circle,
WMB 5105 Atlanta,
nisms underlying AMR is beyond the scope of this
GA 30322, USA Review, but these mechanisms have been extensively
(A. D. Kirk, reviewed elsewhere.8–11
N. A. Turgeon,
N. N. Iwakoshi).
Correspondence to:
A. D. Kirk Competing interests
adkirk@emory.edu The authors declare no competing interests.
B‑cell tolerance of self antigens is established through
multiple mechanisms that take place at various stages of
B‑cell development and differentiation (Figure 1). These
processes are only briefly outlined in this article as they
have been thoroughly discussed elsewhere.12,13
Much of our knowledge of these mechanisms has
been derived from studies of autoreactive B cells. B‑cell
receptors are assembled through a stochastic process of
V(D)J genetic recombination that takes place in devel‑
oping B‑cell precursors. The membrane‑bound anti‑
body moiety of the resulting B‑cell receptors comprises
a large repertoire of randomly selected heavy and light
chains. The receptor diversity achieved via this approach
is enormous, and a substantial number of receptors with
autoreactive potential are produced. Indeed, 20–50% of
B‑cell receptors generated by V(D)J genetic recombina‑
tion are thought to carry an unacceptably high affinity
for self antigens. Consequently, B‑cell maturation incor‑
porates processes that reduce the proportion of auto‑
reactive B cells, thereby helping to prevent autoimmune
diseases.14–17 In general, these processes belong to two
categories: those that eliminate autoreactive B cells in
the central and peripheral lymphoid organs, and those
that prevent activation and differentiation of surviv‑
ing autoreactive B cells (which are generally not as
strongly autoreactive as the B cells eliminated in the
lymphoid organs) into plasma cells during an immune
response. Although the specific mechanisms that deter‑
mine which of these pathways are induced in a par‑
ticular autoreactive B cell are unclear, elimination of
high‑affinity autoreactive receptors tends to involve
clonal deletion and receptor editing, whereas elimina‑
tion of low‑affinity autoreactivity tends to involve
anergy. As a result of these processes, less than 10%
of the immature B cells formed in the bone marrow
actually reach the periphery of the lymphatic system as
transitional B cells.

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Mechanisms in central lymphoid tissues
Clonal deletion
In the primary lymphoid organs, immature B cells that
express strongly autoreactive receptors are induced to die
by interaction with self antigen via ‘clonal deletion’.18 If
stimulation of a B‑cell receptor by a self antigen results in
intracellular signaling of sufficient strength, the immature
B cell rapidly internalizes the autoreactive B‑cell recep‑
tor and temporarily halts its maturation.19–21 If a B cell
that possesses an autoreactive receptor fails to attenuate
or eliminate its autoreactivity, cell death occurs within a
few days, either in the bone marrow or shortly after the
B cell arrives in the periphery of the lymphatic system
as a transitional (or stage T1/T2) B cell (Figure 1).21,22
B‑cell‑receptor‑induced cell death pathways are induced
through increased levels of Bcl2‑like protein 11. This
proapoptotic factor inhibits expression of essential B‑cell
survival proteins of the Bcl2 family.23–25
several other regulatory mechanisms associated with
this arrest of maturation promote self tolerance. Adhesion
molecules responsible for the migration of B cells, such as
l‑selectin and receptors specific for tumor necrosis factor
(TnF) ligand superfamily member 13B (TnFsF13B, also
known as BAFF or Blys), a circulating cytokine required
to sustain the survival of B cells in the periphery, fail to
be induced in persistently autoreactive B cells.21,26
Receptor editing
RAG1 and RAG2, the genes that encode the core
enzymes responsible for V(D)J recombination, con‑
tinue to be expressed in autoreactive B cells that are not
eliminated by clonal deletion. Persistent expression of
RAG genes favors further V(D)J recombination of new
light chains. This process enables B cells that initially
bear an autoreactive receptor to attenuate or overcome
this autoreactivity.27–29
Mechanisms in peripheral lymphoid tissues
Anergy
Autoreactive B cells that elude clonal deletion and recep‑
tor editing can be regulated by clonal anergy, which is
defined as the inability of autoreactive cells to respond
to stimulation by self antigen in the periphery. Anergy
is induced in transitional B cells at stage T1 and T2
and in mature B cells (Figure 1), and is characterized
by molecular changes that reduce the capability of
autoreactive B cells to become activated.30–36
A number of well‑documented intrinsic regulatory
mechanisms are involved in the induction of anergy
in B cells. Among the observed changes that involve
an increase in the threshold of B‑cell activation are a
modulation of the level of the IgM antibody receptor
on the surface of B cells, reduced intracellular signaling
resulting from B‑cell‑receptor stimulation and inhibition
of immunogenic nuclear factor κB signaling pathways.
Continuous B‑cell‑receptor signaling in anergic cells
maintains tolerogenic pathways mediated by eRK and
nFAT signaling by mechanisms that are as yet unclear,
and induces expression of the proapoptotic protein,
Bcl2‑like protein 11.33,37–39
nATuRe ReVIews | NEphRology
© 2010 Macmillan Publishers Limited. All rights reserved
Key points
■ Transplantation tolerance is currently an unattained goal; however, awareness
is increasing that B cells might have an important role in both graft rejection
and tolerance
■ In general, strategies to remove alloantibodies or suppress their production
have met with limited therapeutic success
■ Emerging treatments for antibody-mediated renal allograft rejection need to be
assessed in randomized controlled studies
■ The achievement of sustained transplantation tolerance might require induction
of B-cell tolerance to specific donor antigens
■ To be clinically useful, strategies for inducing B-cell tolerance must integrate
safely into existing immunosuppressive regimens as well as modify the function
and make-up of relevant B-cell subsets
Ignorance
Autoreactive B cells that have a low affinity for systemic,
sequestered or tissue‑specific antigens can bypass clonal
deletion, receptor editing and anergy and persist in the
population of naive B cells. Collectively, these B cells are
described as clonally ignorant and are regulated by a
variety of extrinsic mechanisms, as discussed below.40–42
Mechanisms extrinsic to B cells
A new hypothetical mechanism has emerged in which
transitional B cells in peripheral lymphoid tissues
compete for limited availability of a follicular growth
factor, now identified as TnFsF13B.43–46 In contrast to
clonal deletion in the bone marrow, which is intrinsically
mediated by the B‑cell receptor, the regulation of periph‑
erally located autoreactive B cells is thought to involve a
cell‑extrinsic mechanism that is responsive to homeo‑
stasis of the peripheral B‑cell population. Regulation
of peripheral B‑cell activity during the initiation of an
immune response and in germinal center reactions might
involve antigen availability, competition for develop‑
mental growth factors, T‑cell co‑stimulation and/or
proinflammatory mediators.
Current therapies for AMR
Treatments that address B‑cell‑mediated mechanisms
of graft rejection and induce alloantigen‑specific B‑cell
tolerance might enable renal allograft transplantation
to be performed in sensitized patients. These trans‑
plant recipients are among the most difficult to treat.
Although the T‑cell alloimmune responses of sensitized
patients can be ameliorated with current drug regimens,
their antibody‑dependent effector responses remain
poorly controlled with therapies that target alloantibody
production or induce generalized suppression of
humoral immunity.
Removal of alloantibodies
strategies to halt or suppress alloantibody production
have generally met with limited therapeutic success. In
patients with evidence of alloantibody sensitization before
transplantation (as determined by cellular cross‑match
assays and solid‑phase antibody testing) and in allograft
recipients with evidence of AMR, treatments that remove
alloantibodies have resulted in fair allograft survival.3
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Central lymphoid tissues Peripheral lymphoid tissues

Memory
B cell
Receptor
editing
Pre-B cell T1/2 Mature B cell Differentiation

Plasma
cell
Immature,
self-reactive Self-antigen Tolerance Tolerance
B cell interaction induction induction

Induced cell death Anergic/ignorant Anergic/ignorant

B cell B cell
B-cell
tolerance Central Peripheral

Receptor editing
Clonal deletion

Anergy
Ignorance

Strategies to
■ Mixed chimerism ■ Lymphocyte depletion
induce B-cell
■ Neonatal tolerance ■ Co-stimulation blockade
tolerance
Figure 1 | Physiological mechanisms of B-cell tolerance. Immature autoreactive B cells undergo clonal deletion and
receptor editing in primary lymphoid tissues. When strongly autoreactive B cells interact with self antigens, cell death
rapidly ensues (clonal deletion). Weakly autoreactive B cells that bypass deletion will undergo receptor editing, which
results in removal or attenuation of their autoreactivity. Anergy and ignorance begin in the primary lymphoid tissues but
predominantly occur in secondary lymphoid tissues. Emerging strategies to induce B-cell tolerance in transplantation
involve neonatal tolerance, mixed chimerism, lymphocyte depletion and co-stimulation blockade. Abbreviation: T1/T2,
transitional B cell.

Plasma exchange nonselectively eliminates allo‑
antibodies, but the efficacy of this strategy in improving
allograft survival in these patients is limited by a com‑
pensatory physiological increase in antibody production,
which is induced by hypogammaglobulinemia. Infusions
of nonspecific immunoglobulin (IVIG) might limit this
homeostatic antibody production. such infusions might
also neutralize the de novo production of donor‑specific
antibodies via anti‑idiotypic interactions between vari‑
able regions of IVIG and human leukocyte antigen
(HlA)‑specific antibodies, inhibition of the comple‑
ment system and saturation of the receptors involved in
antibody homeostasis, including IgA Fc receptors and
neonatal IgA Fc receptors.
standard immunosuppressive agents (such as cortico‑
steroids, azathioprine, ciclosporin, mycophenolate
mofetil, tacrolimus and sirolimus) have essentially
no efficacy in treating AMR.3,47 The ineffectiveness of
current immunosuppressive therapies against persistent
alloantibody production, along with current organ‑
allocation policies in the us, has resulted in increased
numbers of alloantibody‑sensitized patients on the renal
transplantation waiting list.
generalized suppression of humoral immunity
Although agents that achieve broad suppression of
humoral immunity, such as rituximab, eculizumab or
bortezomib, have the potential to achieve at least partial
or temporary suppression of alloantibody production
and/or antibody‑mediated effector functions, they do
not achieve stable transplantation tolerance. Importantly,
the safety of the broad inhibition of humoral immunity
achieved by these agents cannot be assumed and requires
confirmation in appropriately designed trials.
Rituximab
Rituximab is a chimeric (mouse and human) CD20‑
specific monoclonal antibody that inhibits B‑cell pro‑
liferation and induces apoptosis by antibody‑dependent
and complement‑dependent cellular cytotoxicity. 48
Administration of rituximab results in a substantial
and rapid depletion of cells that express CD20, includ‑
ing early pre‑B‑stage cells through to cells at late
stages of B‑cell differentiation (but excluding mature
antibody‑secreting plasma cells).
Results from small, noncontrolled studies and case
reports indicate that rituximab helps to reverse AMR
when used as part of a complex multidrug treatment
regimen. Regimens that include rituximab in combina‑
tion with pulsed steroids, plasmapheresis, IVIG, anti‑
lymphocyte globulin and splenectomy have been studied
in patients with AMR that is resistant to conventional
treatment with plasma exchange and IVIG.49–55 such
treatments have resulted in improved graft function, and

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have enabled successful kidney transplantation across
ABo incompatibility barriers.49–55 However, conclusions
on whether these positive results are the consequence
of rituximab’s mode of action cannot be drawn since, in
these studies, this drug was administered concomitantly
with other therapies. Furthermore, CD20 is not expressed
on the surface of mature, antibody‑secreting plasma
cells and the observed beneficial effects of rituximab
administration have been far too rapid to indicate the
presence of a mechanism involving decreased antibody
production or increased clearance. Thus, any therapeutic
benefit of rituximab in patients with AMR probably
involves antigen‑mediated or cytokine‑mediated effects
on B cells. Randomized controlled trials are clearly
necessary to understand how this and other therapies
that deplete B‑cell numbers help to prevent AMR.
Bortezomib
Bortezomib is a selective inhibitor of the 26s protea‑
some.56 In animal studies, bortezomib was effective in
suppressing autoantibody production by nonmalignant
plasma cells.57,58 In these studies, normal plasma cells
were also hypersensitive to proteasome inhibition owing
to their high levels of protein biosynthesis. Proteasome
inhibition by bortezomib eliminates both short‑lived
and long‑lived plasma cells by activating the terminal
unfolded protein response. This response regulates
protein‑biosynthesis homeostasis within the endoplasmic
reticulum (eR); excessive eR stress leads to activation
of cell‑death pathways.59 In vitro data show a signifi‑
cant reduction in the production of antibodies directed
against HlAs following the bortezomib‑induced apop‑
tosis of plasma cells.60 In addition, results from prelimi‑
nary clinical studies suggest some efficacy of bortezomib
in renal transplant recipients with AMR61 and in renal
transplant recipients with high levels of donor‑specific
antibodies before transplantation.62 However, in each of
these studies, bortezomib was administered in combina‑
tion with other desensitizing therapies, which limits the
conclusions that can be drawn about the utility of borte‑
zomib in treating AMR in renal transplant recipients. The
need to evaluate this new therapy in prospective random‑
ized and placebo‑controlled studies was highlighted by
the results of a 2010 study, in which bortezomib alone
did not decrease the levels of donor‑specific antibodies
in sensitized kidney transplant recipients.63
Eculizumab
unlike rituximab and bortezomib, eculizumab does not
act directly on B cells, but instead suppresses antibody‑
mediated cell destruction by preventing activation of the
complement system. eculizumab is a humanized mono‑
clonal antibody with specificity for complement compo‑
nent C5 (C5). Binding of eculizumab to C5 inhibits the
cleavage of C5 into C5a and C5b and thereby prevents
the formation of the membrane‑attack complex.
Although the role of the complement activation on
the pathogenesis of AMR is unclear, episodes of AMR
are generally accompanied by evidence of early comple‑
ment activation as demonstrated by C4d staining of the
nATuRe ReVIews | NEphRology
© 2010 Macmillan Publishers Limited. All rights reserved
peritubular capillaries in renal biopsy specimens.5–7 A
report published in 2009 described a patient who had
severe AMR being successfully treated using eculizumab
in combination with multiple other interventions.64
Another group of investigators have presented their
preliminary experience of eculizumab treatment at the
time of kidney transplantation in a series of cross‑match‑
positive kidney transplant recipients. Results from this
study showed that C5 blockade prevented the develop‑
ment of AMR in patients who developed high levels of
donor‑specific antibodies after transplantation.65 long‑
term, placebo‑controlled studies are needed to evaluate
the efficacy of this novel approach in the prevention
of AMR.
Emerging targets for B‑cell tolerance
B‑cell tolerance seems to be much more difficult to
achieve in humans than in animal models of allograft
transplantation.66–69 This divergence probably results
from species‑specific factors, different exposure to
environmental antigens, different extents of T‑cell and
B‑cell diversity and different frequencies of primed
alloreactive T‑cell and B‑cell precursors owing to hetero‑
logous immunity, to name a few aspects. nonetheless,
observations derived from animal models of allograft
transplantation have served as the foundation for modern
clinical strategies that aim to achieve allograft tolerance
by targeting B cells.
Neonatal tolerance
Immature B cells, such as those of infants, are particu‑
larly susceptible to the induction of tolerance. owen
demonstrated that fraternal twin calves that had a shared
placental circulation permanently accepted transplanted
skin grafts from their twin, whereas inter‑twin skin grafts
were rejected by calves with separate placental circula‑
tions.70 In a subsequent study, allograft tolerance was
induced in mice by fetal infusion of alloantigens, which
might have led to the clonal deletion of B cells with speci‑
ficity for these antigens.71 neonatal tolerance also occurs
naturally in human monochorionic dizygotic twins who
have different blood groups; such twins become tolerant
to each other’s blood group in utero.72
The risk of antibody‑mediated hyperacute rejec‑
tion has generally been thought to preclude ABo‑
incompatible transplantation. However, infants do not
usually produce ABo antibodies until 5–6 months of age,
which represents a window of opportunity during which
ABo‑incompatible transplantation carries a reduced
risk of hyperacute allograft rejection.73 In 1996, a clini‑
cal trial was conducted that included 10 infant recipients
of ABo‑incompatible heart grafts. standard immuno‑
suppression was employed, and no aggressive therapies
to remove donor‑specific antibodies were implemented.74
eight patients survived, and no hyperacute rejections
or problems attributable to ABo incompatibility were
observed. This clinical experience has subsequently
been reproduced.75 The mechanism by which the ABo‑
incompatible heart graft is tolerated seems to involve
deletion of ABo‑specific B cells that persists as long as
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REVIEWS
the ABo‑incompatible heart is present. Interestingly,
this deletion seems to be transient as B cells specific for
the previously mismatched ABo epitope reappeared in
patients who subsequently underwent retransplantation
with an ABo‑compatible heart graft.76 Thus, in contrast
to neonatal tolerance, which seems to involve centrally
based mechanisms, allograft tolerance seems to require
peripheral antigen.
The findings of a 2004 study of the B‑cell repertoire
in infant recipients of ABo‑incompatible heart grafts
suggested that B‑cell tolerance to donor‑specific blood‑
group antigens can develop spontaneously.77 Although
the development of tolerance to donor HlAs was not
demonstrated in these patients, tolerance to these ABo‑
incompatible heart grafts provided the first demonstra‑
tion that neonatal tolerance could be acquired in humans.
ABo‑incompatible transplantation has been attempted
in pediatric candidates for renal and liver transplantation
with some success, although the opportunity for neonatal
transplantation of these organs is uncommon.52,55,78,79
However, in these patients, aggressive therapies were
implemented to remove donor‑specific antibodies and
suppress B‑cell function before and after transplantation.
undoubtedly, increased understanding of the tolerogenic
mechanisms in infants will enable valuable insight to be
gained into possible routes for achieving B‑cell tolerance
to allografts in adult patients.
Chimerism
In contrast to the neonatal chimerism described by
owen in 1945,70 achieving chimerism in adults is chal‑
lenging, largely owing to the immune barrier imposed by
recipient T cells. This barrier can be overcome in small
animal models as bone marrow engraftment has reli‑
ably been shown to induce durable tolerance to the most
immunogenic allografts.
In mixed chimerism, donor and recipient hemato‑
poietic components coexist in primary lymphoid organs,
which facilitates the clonal deletion of T cells and B cells
that react to donor‑specific alloantigens.80–83 Mixed chi‑
merism, in contrast to full chimerism, can generally be
achieved without myeloablation and has the advantage
of preserving the graft recipient’s immunocompetence
while decreasing their risk of graft‑versus‑host disease.
However, current protocols to achieve mixed chimerism
in humans depend on a rigorous early conditioning
regimen that variably includes T‑cell depletion, transient
maintenance immunosuppression and thymic or total
lymphoid irradiation to prevent rejection of the bone‑
marrow graft.84,85 once established, mixed chimerism
of multilineage hematopoietic cells is associated with
lifelong tolerance of T cells and B cells (mediated by
clonal deletion) that enables the acceptance of any donor
allograft without the need for immunosuppression.69
Mixed chimerism has been used clinically to achieve
transplantation tolerance to renal allografts in patients
with multiple myeloma and consequent renal failure.85,86
In these studies, the bone‑marrow donor was fully
HlA‑matched to the recipient. A similar approach,
which involved bone‑marrow transplantation between
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HlA‑haploidentical individuals (that is, between a parent
and a child) has been implemented in a pilot study in
patients without malignant disease. These patients
received haploidentical bone marrow grafts solely to
induce tolerance to a kidney transplant from the same
donor.87 The haploidentical grafts induced transient, very
limited mixed chimerism that was associated with renal
allograft tolerance in four of five treated patients who
were followed for up to 6 years.88 In the fifth patient, the
renal allograft was lost soon after transplantation owing
to acute AMR. Interestingly, two of the graft recipients
who demonstrated transient mixed chimerism devel‑
oped alloantibodies and one of these individuals showed
clinical signs of AMR. This evidence indicates that trans‑
plantation tolerance can coexist with some level of B‑cell
reactivity to donor alloantigens. The relationship between
transient mixed chimerism and B‑cell‑mediated tolerance
to allograft transplantation has not been established.
Rejection of solid‑organ grafts can still occur despite
the presence of mixed hematopoietic chimerism in non‑
human primates, which indicates that the mechanisms
involved in solid‑organ rejection might rely on cell types
that persist despite mixed chimerism.89 The clinical
importance of alloantibody formation in the presence
of apparent T‑cell tolerance to a renal allograft is cur‑
rently unclear, but development of alloantibodies typi‑
cally has a negative prognostic influence in human renal
transplant recipients.90 loss of chimerism in humans
is typically not followed by graft rejection.87 Given the
transient nature of the chimerism achieved, long‑term
transplantation tolerance achieved in human recipients
of HlA‑mismatched kidney transplants combined with
bone‑marrow infusion is unlikely to result from clonal
deletion of B cells alone. In humans, therefore, some of
the other mechanisms of peripheral B‑cell tolerance,
discussed above, might be involved.
lymphocyte depletion
Targeted inhibition and/or depletion of lympho‑
cytes with polyclonal or monoclonal antibodies non‑
specifically reduces the levels of both T‑cell and B‑cell
precursors that are specific for alloantigens. Although
the two most common lymphocyte‑depleting agents,
alemtuzumab and rabbit antithymocyte globulin (ATG)
predominantly deplete T cells, both also deplete B cells.
This approach might, therefore, be a viable induction
strategy for reducing alloantibody titers before trans‑
plantation or to prevent early and late graft rejection. of
note, plasma cells and some B‑cell subsets are resistant
to lymphocyte‑depletion therapy, which might pose an
important barrier to the use of these agents in inducing
allograft tolerance (Figure 2).
lymphocyte depletion might, however, decrease the
risk of allograft rejection. Furthermore, the cell popula‑
tions that re‑emerge after lymphocyte depletion might
be more susceptible than the original populations to
peripheral tolerogenic mechanisms. Consistent with
this hypothesis, lymphocyte depletion has enabled the
reduction of maintenance immunosuppressive therapy
in many allograft recipients, a phenomenon dubbed
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‘prope’ (derived from the latin word for near) tolerance, 1 Abatacept, belatacept 3 Alemtuzumab
which describes incomplete donor‑specific tolerance or (anti-CD52 Ab)

52
tolerance that requires minimal immunosuppression.91

However, the effects of alemtuzumab and rabbit ATG
therapies on B‑cell tolerance have not been evaluated.
Alemtuzumab is a humanized, monoclonal antibody
directed against CD52. This agent induces profound
T‑cell depletion and moderate B‑cell and monocyte
depletion. CD52 is not expressed by plasma cells or
lymphocyte precursors and memory T cells and memory
B cells are refractory to alemtuzumab‑mediated deple‑
tion, which might limit the utility of alemtuzumab to
induce transplantation tolerance.92,93
Induction therapy with alemtuzumab in combina‑
tion with low‑dose immunosuppressive maintenance
therapy has shown promising results in terms of graft
survival in renal transplant recipients. 94–106 However,
despite the proven efficacy of alemtuzumab in decreas‑
ing the number of B cells, therapy with this monoclonal
antibody did not prevent AMR. In fact, treatment with
this agent might be associated with increased rates of
AMR, as demonstrated by C4d staining of peritubular
capillaries, particularly when alemtuzumab is admin‑
istered without a calcineurin inhibitor or to patients
with evidence of prior subclinical sensitization. 96,107
Homeostatic lymphocyte proliferation activates T cells,
and thus might promote T‑cell‑mediated B‑cell anti‑
body production. Phase II trial reports published in
the past 2 years or so revealed that use of alemtuzumab
for the treatment of early relapsing–remitting multiple
sclerosis was associated with the development of auto‑
antibodies, predominantly against thyroid antigens and
blood products, in 30% of patients.108–110 In summary,
the simple concept that partial B‑cell depletion mediated
by alemtuzumab can reduce B‑cell function might be
fundamentally flawed.
T h e p o l y c l o n a l r a b b i t AT G p r e p a r a t i o n ,
Thymoglobulin® , contains antibody molecules with
specificity for many cell surface proteins including
those found on B cells and T cells. Thymoglobulin® as
an induction therapy in renal transplant recipients effec‑
tively reverses early renal transplant rejection. The effect
of Thymoglobulin® on early and late AMR is unclear.
TNfSf13B inhibition
TnFsF13B is a member of the TnF superfamily of pro‑
teins. It is a secreted cytokine that, along with TnFsF13
(formally termed APRIl), interacts with three recep‑
tors expressed on the surface of B cells: TnFRsF13B,
TnFRsF17 and, most importantly, TnFRsF13C (form‑
erly termed TACI, BCMA and the BAFF receptor, respec‑
tively). Integrated signals from both the B‑cell receptors
and members of the TnF ligand superfamily are essen‑
tial for the establishment and maintenance of B‑cell
clones.111,112 Together with B‑cell receptor signaling, TnF‑
ligand‑superfamily signaling transduces survival signals
that determine the proportion of newly formed B cells
that survive to maturity, the longevity of mature primary
B cells, and the differentiation of antigen‑exposed subsets
of B cells.26,46,113 Moreover, substantial depletion of T cells
CD
CD28 B7.1/2 6
MCH CD20 Rituximab
CD4+ TCR (anti-CD20 Ab)
T cell
B cell
D5
2 CD154 CD40 BC
C R Alloantigen
3 2 BAFF-R
Alemtuzumab Anti-CD40 Ab, TACI
(anti-CD52 Ab) anti-CD154 Ab BCMA
4 BR3-Fc
BAFF blockade 5
IVIG
4
Plasmapheresis
Belimumab
BAFF-specific mAb
4
Atacicept (TACI–Ig)
Plasma cell
BAFF and April blockade
Figure 2 | Potential strategies for treating antibody-mediated rejection (AMR).
Dendritic
Abatacept andcell
belatacept (1) are fusion proteins that compete with CD28 for
binding with B7.1 (CD80) or B7.2 (CD86) and block T-cell co-stimulation of B-cell
activation and antibody production. Efforts to target the pathway associated with
the interaction between CD40 and CD154 (2) have been uniformly successful in
controlling humoral responses in animal models. Alemtuzumab (3) is a humanized
mAb directed against CD52 that induces T-cell depletion with moderate depletion
of B cells and monocytes. Members of the TNF superfamily of proteins and of the
TNF superfamily of ligands regulate humoral immunity by controlling B-cell survival
and differentiation. Belimumab and BR3-Fc (4) are TNFSF13B-specific inhibitors.
Atacicept (or TACI–Ig) (4) inhibits both TNFSF13B and TNFSF13. Plasmapheresis
and IVIG (5) enable the nonselective elimination or suppression of alloantibody.
Rituximab (6), a chimeric (mouse and human) CD20-specific mAb, markedly
depletes levels of B lymphocytes. Abbreviations: BAFF-R, BAFF receptor; BCR, B-cell
receptor; BCMA, B-cell maturation antigen; IVIG, intravenous immunoglobulin; mAb,
monoclonal antibody; MHC, major histocompatibility complex; TCR, T-cell receptor;
TNF, tumor necrosis factor; TNFRSF13B, TNF receptor superfamily member 13B;
TNFRSF13C, TNF receptor superfamily member 13C; TNFSF13, TNF ligand
superfamily member 13; TNFSF13B, TNF ligand superfamily member 13B.
and B cells has been associated with increased produc‑
tion of TnFsF13B, perhaps in response to homeostatic
pressure to replace the depleted lymphocytes. Increased
TnFsF13B production might lead to paradoxical B‑cell
activation in the setting of B‑cell lymphopenia.114
since the TnFsF13B‑mediated processes that regulate
B‑cell survival are critical for efficient B‑cell‑mediated
immune responses, members of the TnF ligand super‑
family have emerged as potential key players in the
etiology and treatment of autoimmunity and AMR.
Modulating B‑cell homeostasis and survival via targeting
of TnFsF13B has potentially important implications for
transplantation tolerance. For example, ablation of B‑cell
clones in combination with controlled repopulation
using TnF ligand superfamily inhibitors might facilitate
the induction of transplantation tolerance, because the
newly emerged B cells could undergo induction of tolero‑
genic mechanisms in response to exposure to donor
antigens (Figure 2).
Currently, at least three agents that inhibit the inter‑
action of TnFsF13B with receptors on the surface of
B cells are being developed. Belimumab is a fully human
monoclonal antibody against TnFsF13B. Atacicept is a

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REVIEWS
fusion protein that combines the extracellular ligand‑
binding portion of TnFRsF13B with an Fc region of IgG
to inhibit binding to TnFsF13B and TnFsF13. BR3‑Fc
is a recombinant fusion protein that utilizes the ligand‑
binding portion of TnFRsF13C to prevent its binding to
TnFsF13B (Figure 2).
Co-stimulation blockade
The ‘help’ provided by T cells to antigen‑specific naive
B cells in the form of co‑stimulatory signals and cyto‑
kines is essential to the formation of antibody‑secreting
cells.115,116 Transplant rejection processes mediated by
T cells and by B cells are not, therefore, mutually exclu‑
sive. However, in anamnestic immune responses (which
involve the prompt reappearance of antibodies after
rechallenge with an antigen that had previously induced a
primary immune response) the extent of interdependency
of T‑cell and B‑cell responses decreases.
Co‑stimulatory molecules are cell‑surface receptors
that provide necessary accessory signals at the time
of antigen–receptor interaction to shape the magni‑
tude and character of the subsequent antigen‑specific
response. Blockade of T‑cell co‑stimulatory molecules
has been effectively targeted to achieve T‑cell tolerance
to allograft transplantation, and this approach also pre‑
vents the formation of alloantigen‑specific antibodies
and auto antibodies. 117–120 Most co‑stimulatory mol‑
ecules have been identified on the basis of their influ‑
ence on T cells; B‑cell‑specific co‑stimulatory molecules
have also been identified, but as yet they have not been
used therapeutically.
The receptor–co‑stimulator interaction pathways
CD28–CD80, CD28–CD86 and CD154–CD40 have
been extensively studied in animal models of transplanta‑
tion. The extent to which these pathways influence B‑cell
function through control of T‑cell help is hard to define.
Given that anamnestic responses become decreasingly
susceptible to co‑stimulatory blockade, it is likely that
much of their influence is T‑cell related. nevertheless,
these pathways clearly influence antibody formation and
are involved in the presentation of antigens by B cells to
T cells. Co‑stimulation blockade (that is, blockade of
CD28–B7.1, CD28–B7.2 and/or CD154–CD40 interaction
pathways) has been used in several murine and non‑
human primate models of allograft rejection to induce
tolerogenic mechanisms that are active during peripheral
antigen encounters in graft rejection, either in animals
with an allograft alone or in animals with an allograft and
transfused hematopoietic cells (Figure 2).66,69,121
Abatacept, a fusion protein that competes with CD28
for binding with CD80 (B7.1) and CD86 (B7.2), impedes
the formation of autoantibodies. Belatacept, a derivative
of abatacept with increased affinity for CD80 and CD86,
was developed specifically for use in the organ trans‑
plantation setting, where robust immunosuppression
is required.122,123 Both belatacept and abatacept inhibit
de novo formation of antibodies (including allo‑
antibodies) in non‑primates.119 The results of a random‑
ized clinical trial in renal transplant recipients showed
that patients treated with belatacept have lower rates of
590 | OCTOBER 2010 | vOlumE 6
© 2010 Macmillan Publishers Limited. All rights reserved
positivity for donor‑specific antibodies than patients
treated with ciclosporin.124 efforts to inhibit the pathway
associated with the CD40–CD154 interaction have been
uniformly successful in achieving transplantation toler‑
ance in animal models. Interestingly, despite the clear
role of CD40 stimulation in B‑cell activation and in B‑cell
class switching, blockade of the CD40–CD154 interac‑
tion does not seem to inhibit alloantibody production.125
The reasons for this observation are unclear (Figure 2).
several other B‑cell co‑stimulatory interactions have
been defined, such as the CTlA‑4–ICos ligand inter‑
action, the CD27–CD70 interaction and the CD134–
oX40l interaction.126 The effects of blockade of these
interactions should be tested in rigorous animal models
of allograft transplantation.
Spontaneous renal transplant tolerance
Reports of cases of spontaneous allograft tolerance
demon strate that transplantation tolerance can be
achieved. Generally, these spontaneous phenomena
have been detected in patients who were nonadherent to
immunosuppressive therapy or who required immuno‑
suppression withdrawal owing to complications, both of
which commonly lead to organ rejection. The proportion
of kidney transplant recipients reported as spontane‑
ously tolerant is small (<1% of all renal transplant recipi‑
ents), so performing a systematic mechanistic analysis
in these patients has not been possible. However, a reg‑
istry established by the Immune Tolerance network has
been used to conduct an exploratory mechanistic study.
The investigators identified 25 kidney transplant recipi‑
ents who were considered transplant tolerant, defined
as having stable graft function and having received no
immunosuppression for at least 1 year. An analysis of the
gene‑expression profiles in subsets of peripheral blood
lymphocytes from these patients, from patients with
stable graft function who were on immunosuppression,
and from healthy controls, suggested that allograft toler‑
ance was strongly associated with an increased expres‑
sion of multiple genes involved in B‑cell differentiation.
This subset of B‑cell‑related genes or ‘B‑cell signature’
was consistent with the upregulation of CD20 mes‑
senger RnA in urine sediment cells and the elevated
numbers of naive and transitional B cells as assessed by
flow cytometric analysis in spontaneously transplant‑
tolerant kidney transplant recipients. 127 similarly, a
study from the Indices of Tolerance european union
consortium, which analogously screened 11 opera‑
tionally tolerant kidney transplant recipients, cohorts
of immunosuppressed recipients exhibiting allograft
injury and healthy controls, found that peripheral
blood from tolerant patients contained higher levels
of B‑cell‑related genes than peripheral blood from the
other patients.128 These results suggest a critical role for
B cells in the regulation of the alloimmune response in
transplant‑tolerant kidney graft recipients. In addition,
the results provide a candidate set of genes that may
serve as biomarkers to identify renal transplant recipi‑
ents who may benefit from minimization or withdrawal
of immunosuppression.
www.nature.com/nrneph
 f o C u S o N To l E R A N C E I N T R A N S p l A N TAT I o N

Conclusions will be able to integrate them safely into existing

T‑cell‑mediated allograft rejection is generally well
controlled and understood. However, alloantigen‑
specific B‑cell biology is less well understood than
T‑cell biology, and B‑cell‑mediated humoral immune
responses are only now being recognized as a significant
barrier to developing allograft tolerance. Many thera‑
pies are emerging that have the ability to control B‑cell‑
mediated immune responses. ultimately, the clinical
efficacy of these strategies will depend on their ability
to shape the function and identity of B‑cell subsets,
and whether the research and clinical communities
immunosuppressive regimens.
Review criteria
We searched the MEDLINE database for papers in
English published between 1970 and 2009 (inclusive),
using the terms: “B-cell tolerance”, “antibody-mediated
rejection”, “neonatal tolerance”, “mixed chimerism”,
“alemtuzumab”, “rituximab”, “eculizumab”, “bortezomib”,
“BAFF and transplantation”, “co-stimulation blockade and
transplantation”. The reference lists of retrieved articles
were also searched to identify relevant papers.

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Author contributions
A. D. Kirk, N. A. Turgeon and N. N. Iwakoshi
contributed equally to researching data, discussion of
content, writing, and reviewing/editing the manuscript
before submission.
VoluMe 6 | oCToBeR 2010 | 593