CLINICAL RESEARCH www.jasn.

org

Tolerant Kidney Transplant Patients Produce B Cells

with Regulatory Properties
Mélanie Chesneau,*† Laure Michel,*†‡ Emilie Dugast,*‡ Alexis Chenouard,*† Daniel Baron,*†
Annaïck Pallier,* Justine Durand,*† Faouzi Braza,*† Pierrick Guerif,‡ David-Axel Laplaud,*†‡
Jean-Paul Soulillou,*†‡ Magali Giral,*†‡ Nicolas Degauque,*† Elise Chiffoleau,*† and
Sophie Brouard*†‡
*Joint Research Unit 1064, French Institute of Health and Medical Research, Nantes, France; †Faculty of Medicine,
Nantes University, Nantes, France; and ‡Institute of Transplantation Urology and Nephrology, Nantes University
Hospital, Nantes, France

ABSTRACT
Whereas a B cell–transcriptional profile has been recorded for operationally tolerant kidney graft patients,
the role that B cells have in this tolerance has not been reported. In this study, we analyzed the role of B
cells from operationally tolerant patients, healthy volunteers, and kidney transplant recipients with stable
graft function on T cell suppression. Proliferation, apoptosis, and type I proinflammatory cytokine pro-
duction by effector CD4+CD252 T cells were measured after anti-CD3/anti-CD28 stimulation with or
without autologous B cells. We report that B cells inhibit CD4+CD252 effector T cell response in a dose-
dependent manner. This effect required B cells to interact with T-cell targets and was achieved through a
granzyme B (GzmB)–dependent pathway. Tolerant recipients harbored a higher number of B cells expressing
GzmB and displaying a plasma cell phenotype. Finally, GzmB+ B-cell number was dependent on IL-21 pro-
duction, and B cells from tolerant recipients but not from other patients positively regulated both the number
of IL-21+ T cells and IL-21 production, suggesting a feedback loop in tolerant recipients that increases exces-
sive B cell activation and allows regulation to take place. These data provide insights into the characterization
of B cell–mediated immunoregulation in clinical tolerance and show a potential regulatory effect of B cells on
effector T cells in blood from patients with operationally tolerant kidney grafts.

J Am Soc Nephrol 26: 2588–2598, 2015. doi: 10.1681/ASN.2014040404

Tolerance in transplantation is defined as the main- peripheral B cells has been reproducibly found.9–14
tenance of long-term, good, stable graft function in Several of these B cell markers are being currently
the absence of immunosuppression.1,2 Numerous tested and validated in multicenter studies around
studies have demonstrated that tolerance can easily the world to predict patients who may benefit from
be achieved in rodent models,3–5 including models immunosuppression withdrawal. In addition, some
for renal transplant.6 However, while it remains of these B cell markers are being examined for their
rare in human renal transplant it does exist, current involvement in the tolerance process; however, to
estimates report roughly a hundred cases of opera- date, none have been shown to have a role in tolerance
tional tolerance, mainly patients not compliant
with their immunosuppressive regimens.1,2,7 These Received April 24, 2014. Accepted December 11, 2014.
patients, defined as “operationally tolerant,” are Published online ahead of print. Publication date available at
healthy, do not exhibit more infections or malig- www.jasn.org.
nancies than healthy volunteers (HVs), and do not
Correspondence: Dr. Sophie Brouard, Joint Research Unit 1064,
display clinical evidence of immune incompetence.2,8 Institute of Transplantation Urology and Nephrology, Nantes
Specific patterns have been associated with this op- University Hospital, 30 Bd Jean Monnet, 44093 Nantes Cedex 01,
France. Email: [email protected]
erational tolerance; in particular, a B cell transcrip-
tional signature that correlates with an increase in Copyright © 2015 by the American Society of Nephrology

2588 ISSN : 1046-6673/2610-2588 J Am Soc Nephrol 26: 2588–2598, 2015

 www.jasn.org CLINICAL RESEARCH

induction or maintenance, and whether B cells are involved, or B cells and a 1:2 T cell/B cell ratio were used in all of the experi-
even have a potential role in tolerance, remains to be determined. ments. The addition of prestimulated B cells to the coculture
In transplantation, B cells are mainly known for their induces a significant increase in CD4+CD252 T cell apoptosis
capacity to differentiate into plasma cells and produce anti- in the three groups (Figure 1C). Interestingly, no difference was
bodies that may be deleterious for the graft.15,16 However, B observed in apoptosis levels between cell trace+ and cell trace–
cells also have antibody-independent functions. They are able T cells, confirming that the increase in apoptosis was not due to
to produce cytokines and to present antigens, and thus to inhibition of T cell proliferation (data not shown). Type I helper
initiate or maintain an immune response.17,18 More recently, T cell (Th1) proinflammatory cytokines (IFN-g and TNF-a)
populations of regulatory B cells (Bregs) able to dampen the were analyzed using intracellular staining after 3 days of co-
immune response have been highlighted as a “driving force” in culture. IFN-g T cell production was slightly lower when
autoimmune diseases, cancers, and transplantation. 19–23 prestimulated B cells from HVs were added to the culture,
However, their nature, origin, phenotype, and mode of action but this was due to a slightly higher level of IFN-g production by
in humans remain little known. CD4+CD252 T cells from HVs only (Figure 1D). TNF-a pro-
We previously reported that B cells from tolerant patients duction by T cells from the three groups of patients was un-
(TOLs) do not fully differentiate into plasma cells and that, changed when prestimulated B cells were added to the culture
during their differentiation, B cells from tolerant recipients (Figure 1E). Representative pictures of IFN-g and TNF-a pro-
produce higher levels of IL-10,24 suggesting that an imbalance duction by T cells are displayed in Figure 1, F and G. Altogether,
between a lower number of deleterious plasma cells and a these data show that B cells from HVs, transplant TOLs, and STAs
higher level of Bregs producing IL-10 may exist in these pa- all inhibit T cell proliferation and induce T cell apoptosis but have
tients. In this article, we investigate the role of B cells in blood no effect on Th1 proinflammatory cytokine production.
from this cohort of operationally TOLs, from patients with
stable graft function under classic immunosuppression B Cell Inhibitory Effect on T Cells Is Dependent to
(STAs), and from HVs. We report that tolerant recipients GzmB and Is Contact Dependent
exhibit a higher number of IL-21–dependent peripheral B cells Having previously reported higher production of IL-10 by
that express granzyme B (GzmB), display a specific phenotype, B cells from tolerant recipients during the differentiation process
and exhibit a contact- and GzmB-dependent, IL-10– and TGF- in vitro, as well as B cells having been shown to mainly display
b–independent inhibition of effector T cell response. These regulatory properties through IL-10, we decided to assess the
results provide novel insights into the characterization of B role of IL-10 in our model. We looked at the frequency of
cell–mediated immunoregulation in tolerance in the clinic. IL-10–expressing B cells and the level of IL-10 expression by
these B cells after 48 hours of CD40L and oligodeoxynucleotide
(ODN) stimulation. As expected, although the resting B10
RESULTS level was low, a significant and substantial increase in the fre-
quency of B10 cells was found after activation (Figure 2A). No
CpG-CD40 Prestimulated B Cells Inhibit CD4+CD252 difference was observed in the frequency of B10 cells and in the
T Cell Proliferation relative amount of IL-10 expressed by B cells between the three
Human B cells have been shown to exhibit regulatory activity groups of individuals (Figure 2, B and C). To assess the role of
through the inhibition of various cell types, in particular IL-10 in the coculture assay, we blocked its effect using anti–
through the inhibition of T cells. CD4+CD252 T cells were IL-10 antibody. We found that the blockade of IL-10 does not
cocultured with CpG-CD40 stimulated or unstimulated hinder the inhibitory effect of B cells on effector T cell pro-
autologous B cells, after polyclonal anti-CD3 and anti- liferation (Figure 3A). Because other cytokines have been
CD28 activation, for 3 days and T cell proliferation was shown to play a role in the function of suppressive B cell pop-
then analyzed, using CellTrace Violet staining. We found ulations, TGF-b and GzmB were similarly blocked by adding
that prestimulated B cells from HVs, TOLs, and STAs signif- anti–TGF-b antibody and anti-GzmB peptide to the coculture
icantly inhibit autologous CD4+CD252 T cell proliferation at day 0. The blockade of TGF-b did not hinder the inhibitory
(Figure 1A), whereas unstimulated B cells have a lesser effect effect of B cells on T cell proliferation (Figure 3B). However,
(Figure 1B). No difference was found in the number of total B for the three groups of patients, the addition of anti-GzmB
cells and GzmB+ B cells or for B cell inhibition between men peptide to the coculture significantly affects the suppressive
and women. effect of B cells on autologous CD4+CD252 T cell proliferation
(Figure 3C), whereas GzmB inhibitor has no effect on T cell
CpG-CD40 Prestimulated B Cells Induce T Cell proliferation in the absence of B cells (Figure 3D).
Apoptosis But Have No Effect on Proinflammatory Because the inhibitory functions of B cells involving GzmB
Cytokine Production have been shown to act through a direct interaction of B cells
Using Annexin V staining, apoptosis of CD4+CD252 T cells with their target,25,26 transwell cocultures were performed to
was measured at day 3 after anti-CD3/anti-CD28 activation determine whether contact was required by B cells to inhibit
and addition of prestimulated B cells to the culture. Prestimulated T cell proliferation. As shown in Figure 3E, the inhibitory B cell

J Am Soc Nephrol 26: 2588–2598, 2015 B Cell Regulation in Transplant Tolerance 2589
 CLINICAL RESEARCH www.jasn.org

effect disappeared when B and T cells were

cultured in transwell, demonstrating that
T cell–B cell interaction is necessary for the
inhibitory B cell function. Altogether, these
data show that B cells inhibit T cell prolifer-
ation via a GzmB pathway and depend on
contact between the B and T cells.

Tolerant Recipients Have a Higher

Number of B Cells, Which Act in a
Dose-Dependent Manner and
Express GzmB
On a cell-by-cell basis, B cells from TOLs
added to the coculture have the same ability
as B cells from HVs and STAs to regulate
autologous effector T cell proliferation in a
contact- and GzmB-dependent manner
(Figure 3, C and E). We previously reported
on a higher number of total B cells in blood
from TOLs.10,12,24 Here we found that the
absolute value of GzmB-producing B cells
was significantly higher in TOLs compared
with HVs and STAs (P,0.05; Figure 4A)
and the percentage of GzmB+ B cells is di-
rectly correlated with effector CD4+CD252
T cell proliferation inhibition (Figure 4B).
Altogether, these data show that tolerant re-
cipients have a higher number of peripheral
B cells and GzmB+ B cells that are able to
inhibit T cell response through a contact-
and GzmB-dependent pathway and in a
dose-dependent manner.

Resting and Stimulated GzmB-

Expressing B Cells Display a CD5+
CD27+CD138+ Phenotype
Figure 1. CpG-CD40 prestimulated B cells inhibit CD4+CD252 T cell proliferation The GzmB+ B cell phenotype was analyzed
and induce T cell apoptosis without effect on type Th1 proinflammatory cytokine
by flow cytometry before and after 3 days of
production. Effector T cell proliferation is followed by CellTrace Violet staining after
3 days activated with anti-CD3/anti-CD28 and cocultured with unstimulated B cells
coculture using CD19, CD20, CD138, CD38,
or B cells stimulated with CD40L and ODN 24 hours before the coculture. (A) Pro- CD27, CD24, CD5, CD1d, IgD, IgG, and
portion of T cell proliferation inhibition when cocultured with or without prestimu- IgM cell surface markers. Unstimulated
lated B cells in HVs (n=17), TOLs (n=12), and STAs (n=17) (mean6SEM; GzmB+ B cells express a higher level of
****P,0.001). (B) Representative histograms of proliferation of T cells: alone, with CD138, CD27, CD5, and CD38, and fewer
unstimulated B cells, and with stimulated B cells from HVs. Apoptosis of effectors T IgD markers compared with unstimulated
cells is analyzed by Annexin V staining after 3 days of activation with anti-CD3/anti- GzmB2 B cells (Figure 4C). After stimula-
CD28 and coculture with B cells prestimulated with CD40L and ODN 24 hours before tion, GzmB+ B cells have lower CD38 ex-
the coculture. (C) Percentage of Annexin V T cells activated and cocultured with or pression compared with GzmB2 B cells
without stimulated B cells in HVs (n=9), TOLs (n=10), and STAs (n=12) (mean6SEM; (Figure 4D). Resting GzmB+ B cells repre-
*P,0.05; **P,0.01). Secretion of IFN-g and TNF-a is analyzed by intracellular
sented around 2.7%61.45% of total B cells
staining in effector T cells after 3 days of coculture with B cells and anti-CD3/anti-
from TOLs. Twenty-four hours of activation
CD28 activation. (D) Percentage of IFN-g + T cells activated and cocultured with or
without B cells in HVs (n=17), TOLs (n=12), and STAs (n=14) (mean6SEM; *P,0.05). with CD40L and ADN following by three days
(E) Percentage of TNF-a+ T cells activated and cocultured with or without B cells in of coculture with activated effector T cells
HVs (n=17), TOLs (n=12), and STAs (n=14) (mean6SEM). (F and G) Representative greatly increases the expression of GzmB
dot plots of the secretion of IFN-g and TNF-a by T cells activated and cocultured (20%62.5% after activation) (Supplemental
with or without B cells from HVs. Figure 1, A and B). Altogether, these data

2590 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2588–2598, 2015
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These data clearly show that B cells are not

intrinsically different between the groups
of patients and that, at least for these se-
lected genes, the signature of B cells in
blood from tolerant recipients is mainly
the result of a higher number of B cells in
PBMCs (Supplemental Figure 2).

B Cells from Tolerant Recipients

Regulate IL-21–Producing T Cell
Levels; IL-21 Production by T Cells
Regulates the Number of GZMB+ B
cells
GzmB-expressing B cells have been shown
to depend on T cell–produced IL-21, and
IL-21 is also an important factor in B cell
homeostasis. We found that in TOL stim-
ulation with anti-CD3/anti-CD28 of CD4+
CD252 T cells is associated with a lower
number of IL-21–producing T cells
compared with HVs and STAs (P,0.05).
However, when B cells are added to the co-
culture, IL-21–producing T cells signifi-
Figure 2. IL-10+ B cells and IL-10 secretion after 48-hour stimulation with CD40L/ cantly increased in TOLs (P,0.01),
ODN. IL-10 expression was analyzed on B cells after 48-hour stimulation of PBMCs whereas no difference, or even decreased
with CD40L and ODN. (A) Representative dot plot of IL-10 secretion in resting, IL-21 production, was observed in HVs
stimulated B cells, and stimulated B cells staining with isotype control. (B) Percentage and STAs (Figure 5, A and B). This corre-
of IL-10+ B cells before and after activation in HVs (n=7), TOLs (n=9), and STAs (n=10) lates with a higher production of IL-21 by
(mean6SEM; ***P,0.001). (C) Mean fluorescence intensity from IL-10 stimulated T cells stimulated by B cells from TOLs
B cells in HVs (n=7), TOLs (n=9), and STAs (n=10). compared with those from HVs and STAs
(P,0.05) (Figure 5C). Altogether, these
data show that B cells from TOLs regulate
show that tolerant recipients have a higher number of GzmB- the number of IL-21+ T cells and IL-21 production in vitro.
expressing B cells with a CD138+CD27+CD5+CD38+IgD2 Finally, to assess the role of IL-21 on GzmB-expressing B cells,
phenotype. we blocked its effect using anti–IL-21 antibody in the coculture
assay. Increasing doses of anti–IL-21 (0, 2, 4, 6, and 8 mg/ml)
B Cell Regulatory Transcriptional Profile Is Not significantly decreased the number of GzmB-expressing B cells
Different in TOLs, HVs, and STAs (Figure 5D).
We investigated the expression of 60 markers selected either
for their participation in the regulation of B cell functions
(Supplemental Figure 2, A and B),26 or their implication in a DISCUSSION
tolerance-related B signature (Supplemental Figure 2, C and
D)27 in purified and isolated B cells or PBMCs from samples B cells may have a dual effect, acting as a driver and as a regulator
from 32 individuals (10 TOLs, 10 HVs, and 12 STAs). Among of the immune system.15–18,28–32 We recently showed that tol-
these genes, only CD38 and CD1D exhibited a significant dif- erant recipients are characterized by a higher number of cir-
ference between STAs and HVs in B cells (Supplemental Figure culating B cells,10,12 a defect in terminal differentiation,24
2A), whereas more than half were different in PBMCs (Sup- and a B cell–transcriptional profile with overexpression of
plemental Figure 2B). The second subset (Supplemental Fig- molecules associated with regulation.9,12 In animal models,
ure 2, C and D) includes a panel of 35 markers previously that B cells have a role in tolerance is clearly suggested by
linked to tolerance across blood transcriptomic studies and the accumulation of B cells and formation of germinal centers
preferentially expressed in B cell subsets for 69% of them.27 in tolerant allografts and the ability of B cells to prolong graft
Only two of these markers were different in B cells (EBF1 survival.33 However, the role and nature of Bregs in the mod-
between TOLs and HVs; PLEKHG1 between STAs and HVs) ulation of immune response in alloimmunity and in tolerance
(Supplemental Figure 2D), whereas all of them were different in humans remains unclear. Although tolerant kidney reci-
between TOLs and STAs in PBMCs (Supplemental Figure 2D). pients clearly display a strong B-cell signature, with a higher

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 CLINICAL RESEARCH www.jasn.org

capacity to activate CD40-CD40L signal-

ing, phosphorylate STAT3, and preserve B
cell compartment diversity, suggesting
a role for these B cells in tolerance.14 In
this article, we explore the potential regu-
latory role of B cells in such patients. Be-
cause of the difficulty in identifying a
unique Bregs population with a unique
phenotype in humans, we analyzed the in
vitro suppressive properties of B cells over-
all. We report a higher number of B cells
with dose-dependent suppressive proper-
ties in blood from patients with a tolerant
kidney graft. The inhibitory effect of B cells
is dependent on GzmB and on the interac-
tion of B cells with their T cell targets.
Much evidence suggests that activation is
instrumental in Bregs activity.35 In this ar-
ticle, we show that prestimulated B cells are
more efficient at suppressing effector T cell
proliferation than nonstimulated B cells, a
result in accordance with previous data
showing that maturation and B cell activa-
tion were important parameters in this pro-
cess.28,30–32,36 It may be a little counterintu-
itive to say that B cells need such activation
to regulate the immune system because ac-
tivated B cells are usually associated with
stimulatory activity on other cell types.
However, this has been extensively dis-
cussed and can be explained as the partici-
pation of B cells in a general feedback loop
that both induces an immune process and
prevents excessive inflammation or un-
Figure 3. Regulation of effector T cell proliferation by B cells is contact and GzmB wanted autoaggressive T cell response.37
dependent. (A–C) T cell proliferation inhibition in HVs (n=12), TOLs (n=10), and STAs Moreover, we clearly show that the inhibi-
(n=7) when T cells are activated with anti-CD3/anti-CD8 and cocultured with B cells with tory effect of B cells is dependent on GzmB,
or without anti–IL-10 blocking antibody (A), with or without anti–TGF-b blocking anti- the expression of which occurs in resting
body in HVs (n=10), TOLs (n=8), and STAs (n=6) (B), or with or without GzmB inhibitor B cells in vivo and is increased in stimulated
peptide in HVs (n=9), TOLs (n=9), and STAs (n=14). (D and E) Representative histograms
B cells. Interestingly, as already described for
of T cell proliferation with or without GzmB inhibitor peptide (D) and when T and B cells
the pro-B10 regulatory cell population, this
are cocultured in transwell (E) in HVs (n=6), TOLs (n=4), and STAs (n=3) (mean6SEM;
*P,0.05; **P,0.01; ***P,0.001).
leads to the conclusion that B cell–mediated
regulation is probably inducible and that
the effect of B cells is greatly influenced by
the nature of the microenvironment.35,36,38–40
number of B cells, their potential role in establishing and/or The expression of IL-10 has been shown to be a frequent
maintaining tolerance remains to be determined. characteristic of Bregs.41–43 Such cells, referred to as B10,32,44
It is tempting to speculate that this increase in B cells with a are involved in the initiation, the onset, and the severity of
specific inhibitory profile may be linked to the tolerance of various autoimmune diseases, and in transplantation.45–47 We
these rare patients. Haynes et al. recently proposed an indirect show here that the suppressive properties of B cells are not
pathway model in which a decrease in immunosuppression is dependent on IL-10. Anti–IL-10, used at doses able to effi-
associated with an increased number of indirect pathway reg- ciently block an IL-10 T cell–dependent response (data not
ulatory T cells (Tregs) and of B cells, possibly Bregs, but the shown), does not diminish the inhibitory effect of B cells on T
mechanisms have not been established.34 Silva et al. showed cell proliferation. These data are in agreement with the work of
that peripheral B cells from tolerant recipients maintain the Deng et al., who reported that anti-CD45RB treatment

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and plasmacytoid dendritic cells.55 GzmB

mediates T cell apoptosis and T cell prolif-
eration suppression, independently of the
perforin component.55 In this work, the
fact that a blockade of GzmB with anti-
GzmB induces a diminishment of the
B cell inhibitory effect indicates that GzmB
is functional. Interestingly, we found that
B cells not only inhibit T cell proliferation,
but also induce their apoptosis. We did not
find any involvement of the Fas-FasL path-
way in this process (data not shown), but
alternative pathways have been described
as inducing apoptosis, and GzmB is one
of them.56 These data suggest a pivotal
role for GzmB in regulation by B cells in
our patients through cell death induction
and T cell proliferation inhibition, a mech-
anism already described for Tregs after
polyclonal and antigen-specific activa-
tion.54 Interestingly, we found that B cells
had no effect on TNF-a and IFN-g pro-
Figure 4. Tolerant recipients have a higher number of B cells expressing GzmB and act duction. Iwata et al. reported that IFN-g
in a dose-dependent manner. (A) Number of GZMB+ B cells per microliter of blood and TNF-a production was dependant
from HVs (n=6), TOLs (n=6), and STAs (n=6) (mean6SEM; *P,0.05). (B) Linear re- from IL-10. 46 This is reinforced by the
gression of percentage of GzmB+ B cells and percentage of inhibition of CD4+CD252 data from Lemoine et al., who reported
T cell proliferation (P,0.04). (C) Representative histograms for CD138, CD27, CD5, that regulation of T cell proliferation was
CD38, CD24, CD1d, IgD, IgG, and IgM expression within the GzmB+ (thin line) and distinct from the differentiation of T cells
GzmB2 (thick line) in CD19+ B cells before stimulation. (D) Representative histograms
into Th1 cells that depend on the produc-
for CD38 and CD138 within the GzmB+ (thin line) and GzmB2 (thick line) in CD19+
tion of IL-10.42 These observations are in
B cells after stimulation.
accordance with ours and particularly re-
inforce our finding that IL-10 is not in-
induces strong and antigen-specific tolerance, which is depen- volved in B cell regulation in our present situation. To our
dent on the presence of B lymphocytes and is independent knowledge, nothing has yet been reported on the effect of
from IL-10. In this model, IL-10 clearly counter-regulates tol- GzmB on IFN-g and/or TNF-a production by B cells.
erance induction, and even exerts a negative effect and causes Several microenvironmental factors have been shown to be
histologic lesions of rejection.48 The same observations have instrumental in B cell homeostasis. These factors include
been reported in other models.49 Interestingly, it has been molecules such as IL-21. 26,53 We first demonstrated that
reported that B cells from patients with chronic antibody- GzmB-expressing B cells are dependent on IL-21. Increasing
mediated rejection have a defect in suppressive properties,50 in doses of anti–IL-21 decrease the number of GzmB-expressing
contrast with B cells from STAs, which inhibit T cell prolifer- B cells in coculture. Moreover, when B cells are added to the
ation and induce Treg generation through a TGF-b– and IDO– coculture, IL-21–producing T cells increase significantly and
dependent pathway (personal communication, Nouël et al. Ann there is greater production of IL-21 by T cells from TOLs.
Rheum Dis abstract A8.32, 2014). In our situation, blocking IL-21 is a key cytokine for GzmB gene transcription,26,57
TGF-b has no effect on the suppressive function of B cells, sug- which suggests that the increase in GzmB+ B cells in blood
gesting that TGF-b is not involved in B cell regulation either. from tolerant recipients may be due to a direct effect of IL-21.
Interestingly, other regulation pathways have been shown to These data are reinforced by the effective STAT3 phosphory-
be involved in B cell regulatory activity. We found that GzmB lation in B cells from tolerant recipients,14 a key signal for the
blockade diminishes the B cell inhibitory effect and restores generation of GzmB activated B cells.57 Finally, the fact that
T cell proliferation. This is associated with a higher number of tolerant recipients have a lower level of IL-21+ CD4+ T cells,
GzmB-producing B cells in blood from tolerant recipients and and that B cells from these patients alone (and not HVs and
B cells that act in a contact- and a dose-dependent manner. STAs) increase IL-21 production by CD4+ T cells, strongly
GzmB is a 32-kD serine protease mainly known as a component suggests a negative feedback loop in these patients, increasing
of the cytotoxic granule of T cells and natural killer cells51,52 but excessive B cell activation and inflammation and allowing reg-
is also produced by other cell types, such as B cells,53 Tregs,54 ulation to take place.

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find that GzmB+ B cells express a specific

phenotype with upregulation of CD5,
CD27, CD38, and CD138 markers. This is
in accordance with our previous data
reporting a higher level of memory CD27+
B cells and CD5+ B cells in blood from tol-
erant recipients.12 These data are also in
accordance with the fact that CD5+ B cells
constitutively express GzmB in an IL-21–
dependent pathway59 and that CD5+ B cells
express higher levels of IL-21–R than CD52
B cells.26 We also found that these cells
express CD138 and CD38 and are IgD2,
markers expressed by B cells secreting
IL-10 and IL-35 and involved in suppression
mechanisms,60 and also associated with
plasma cell maturation. 61 Interestingly,
CD138 has also been shown to be associated
with GzmB production, regulatory activity,
and IL-21 dependence.57
In conclusion, we have demonstrated
that TOLs have a higher absolute number of
GzmB+ B cells with a plasma cell2like phe-
notype and with dose-dependent suppres-
sive properties through the GzmB pathway.
B cells decrease T cell proliferation and in-
duce their apoptosis, two processes that may
Figure 5. B cells from tolerant recipients regulate IL-21–producing T cells levels and
IL-21 production by T cells. (A) Dot plot of the secretion of IL-21 by activated
contribute to a tolerogeneic environment.
CD4 CD25 T cells at day 3 of coculture with stimulated B cells. (B) Percentage of GzmB-producing B cells are under the con-
+ 2

IL-21+ T cells at day 3 of activation and coculture with or without stimulated B cells in trol of IL-21. The fact that there is more
HVs (n=10), TOLs (n=12), and STAs (n=14) (*P,0.05; **P,0.01). (C) Concentration of IL-21 in vitro in TOLs after B cell stimulation
IL-21 (pg/ml) in supernatants after 3 days of coculture, T cell coculture with B cells correlates with a greater number of IL-21–
(mean6SEM; *P,0.05). (D) Percentage of GzmB+ B cells normalized on B cells plus dependent circulating GzmBs and a higher
T cells alone after increasing doses of IL-21 blocking molecule is added to coculture inhibitory effect of B cells, which may act as
(0, 2, 4, 6, and 8 mg/ml) (*P,0.05; ***P,0.01). pro-B10 that need to be activated to display
an increased effect. In previous articles, we
reported on a decreased number of circulat-
Bregs are able to control the immune response, but an ex- ing plasma cells and we showed that B cells from tolerant recip-
cessive reaction from these cells may also promote tumor cell ients do not fully differentiate into plasmablasts and are more
growth or chronic infection.26 We hypothesize that this fine- sensitive to apoptosis.12,20 Here again, this is in accordance with
tuning of regulation by B cells and IL-21 production by T cells the fact that in vitro unstimulated T cells from TOLs produce less
might be a key factor in tolerance maintenance. IL-21, a molecule that directly acts on B cell differentiation.62,63
We show that TOLs have more B cells in absolute value with We hypothesize that these properties may contribute to a favor-
regulatory properties, but on a cell-per-cell basis, their B cells able tolerogeneic environment and favorable inversion of the B
have the same suppressive activity as B cells from STAs and effector–plasma cell/Bregs balance in these patients and their
HVs. These results are supported by transcriptional analysis lower antibody production. These data support a role for B cells
that shows that the B cells are not intrinsically different between in patients with operational tolerance. They also raise the ques-
the groups of patients and that, at least for these selected genes, tion of whether it would be possible to trigger an ex vivo or in
the B cell signature in blood from tolerant recipients’ results vivo increase to encourage any potential therapeutic effects, such
mostly from a higher number of B cells in PBMCs. It is not as counteracting the alloimmune response or even promoting
surprising that the greater suppressive effect in blood of tol- tolerance. These data also question the effectiveness of de-
erant recipients is due to a higher number of circulating GzmB+ pleting B cells in order to control antibody-mediated rejec-
cells, if we consider that clinical outcomes in numerous sit- tion, rather than developing new strategies to maintain the
uations are mainly driven by the quantity of infiltrating and fragile balance between the negative and beneficial effects of
circulating cells, more than their quality.58 Interestingly, we B cells in transplantation.

2594 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2588–2598, 2015
 www.jasn.org CLINICAL RESEARCH

CONCISE METHODS detection of intracellular IL-10, staining was performed using anti–
IL-10-phycoérythrine (PE) (clone JES3-9D7; BD Biosciences). Appro-
Patients and HVs priate PE-conjugated isotype controls were used for gate setting for
Forty-one patients took part in the study and signed informed consent as IL-10 expression. Unstimulated B cells were stained and used as a
follows: (1) TOLs with stable kidney graft function (creatinemia,150 control for the gating strategy.
mmol/L and proteinuria,1 g/24 h) in the absence of immunosuppres-
sion for at least 1 year (n=12) except for 2 who have creatinemia.150 B Cell Functional Assays: B and T Cell Purification
mmol/L620% but were stable over time, (2) STAs with stable kidney PBMCs were obtained after Ficoll density centrifugation (Sigma-
graft function (creatinemia,150 mmol/L and proteinuria,1 g/24 h) Aldrich) of fresh blood samples. B cells were purified by negative
for at least 3 years under standard immunosuppression (calcineurin selection using a Human B Cell Isolation Kit II and an autoMACS PRO
inhibitors and corticosteroids) (n=17), and (3) HVs without patholo- Separator, with purity .95% (Miltenyi Biotech, Gladbach, Germany)
gies or infectious episodes in the previous 6 months (n=17) (Table 1). and stimulated for 24 hours with CD40L (1 mg/ml) and CpG-ODN
(10 mg/ml) in a 96-well U-bottom plate at a concentration of 106
Cell Culture cells/ml. As a control, a proportion of PBMCs were kept at 4°C in
PBMCs were stimulated at 23106 cells/ml for 48 hours in complete complete medium for 24 hours. B cells were purified using the same
RPMI 1640 (Sigma-Aldrich, St. Louis, MO) containing L-glutamine, technique, without stimulation. CD4+CD252 responding T cells
penicillin/streptomycin (Life Technologies, Carlsbad, CA), and 10% were purified by negative selection using successively CD4+ T Cell
FCS (Lonza, Verviers, Belgium) in 96-well plates (Nunc, Langensel- Isolation Kit II and CD25+ Microbeads II (Miltenyi Biotech), accord-
bold, Germany) at 37°C, 5% CO2. Stimulation was performed using ing to the manufacturer’s instructions with a purity .95%.
CpG oligonucleotide (ODN 2006, 10 mg/ml; InvivoGen, San Diego, CA)
and recombinant human soluble CD40L (R&D Systems, Minneapolis, Coculture Experiments
MN). PMA (250 ng/ml), ionomycin (1 mg/ml), and brefeldin-A Coculture assays were performed for 72 hours by adding 13105 au-
(10 mg/ml) (Sigma-Aldrich) were added for the last 5 hours of the tologous prestimulated B cells or unstimulated B cells to 0.53105
B-cell culture. As a control, PBMCs were cultured in resting condi- CD4+CD252 responding T cells, stimulated with anti-CD3 and
tions for 48 hours. anti-CD28.2 dynabeads (at a 1:1 ratio of dynabeads/T cells) (Invitrogen,
Oslo, Norway). After 72 hours of coculture, brefeldin-A was
Analyses of IL-10 Production by Stimulated B Cells added at 10 mg/ml for 4 hours. Viability of T and B cells was checked
After 48 hours of culture, viability staining was performed using the by 49,6-diamidino-2-phenylindole staining. The proliferation of
aqua Live/Dead cell staining kit (Invitrogen/Life Technologies). B CD4+CD252 responding T cells was measured after staining with
lymphocytes were stained with anti–CD19-PC7 (BD Biosciences, San CellTrace Violet (Invitrogen). T cell IFN-g, IL-21, and TNF-a secretion
Diego, CA), washed, fixed, and permeabilized using a permeabilization/ was measured after permeabilization of responding T cells and staining
fixation kit (BD Biosciences). Fcg receptor inhibitor (eBiosciences, with anti–CD4-PE (BD Biosciences), anti–IFN-g-allophycocyanin
San Diego, CA) was used to avoid nonspecific staining. For (APC) and anti–TNF-a-fluorescéine isothiocyanate (FITC) (BD

Table 1. Summary of clinical and demographic characteristic of patients and HVs

Time Time between
Donor (LD
Patient Age Sex HLA between Creatinemia Proteinuria Immunosuppression
versus
Group (yr) (F/M) Mismatches (n) Graft and (mmol/L) (g/24 h) Withdrawal and
NLV)
Analysis (mo) Analysis (yr)
HV (n=17) 8/17
Median 46.5
SD 10.8
Minimum 27.0
Maximum 61.0
TOL (n=12) 6/12 4/12
Median 58.0 3.0 220.6 90.0 0.1 10.5
SD 15.8 1.9 94.9 64.7 0.2 5.5
Minimum 31.4 0.0 71.4 66.0 0.0 1.0
Maximum 85.3 4.0 385.3 280.0 0.6 19.0
STA (n=17) 5/17 1/17
Median 57.6 4.0 120.4 124.0 0.1
SD 10.8 1.4 37.6 29.1 0.1
Minimum 40.4 1.0 66.4 74.0 0.0
Maximum 74,9 5.0 192.5 175.0 0.2
F, female; M, male; LD, living donor; NLV, nonliving donor.

J Am Soc Nephrol 26: 2588–2598, 2015 B Cell Regulation in Transplant Tolerance 2595
 CLINICAL RESEARCH www.jasn.org

Biosciences). For measurement of T cell apoptosis, cells were stained ACKNOWLEDGMENTS

with CD4-APC, CD19-PE-C7, and Annexin V-APC (BD Biosciences).
To investigate whether B cells have a dose-dependent effect on T cells, We thank the patients and their families, whose trust, support, and
the number of B cells was progressively increased in the coculture cooperation were essential for the collection of the data used in this
(50,000, 100,000, and 200,000), whereas the number of 50,000 T cells study. We also thank G. Blancho, Dr. D. Cantarovitch, J. Dantal,
remained fixed. Dr. S. Ferrari-Lacraz, K. Hadaya, M. Hourmant, Dr. B. Hurault de
Ligny, Dr. G. Lefrancois, C. Legendre, Dr. B. Le Mauff, Dr. H. Le
Coculture Experiments Blockade Monies De Sagazan, Dr. A. Meurette, Dr. M. Rabant, J.F. Subra, Dr. J.
These coculture assays were performed under the same conditions Sayegh, Dr. A. Testa, Dr. F. Villemain, and Dr. J. Zuber for their help in
using transwell polycarbonate inserts (0.4 mm; Corning, Inc.) or using this study.
anti–IL-10 (BD Biosciences), anti–TGF-b1 (Abcam, Inc., Cambridge, This work was carried out with the support of CENTAURE and
UK), or anti-GzmB, a peptide that irreversibly inhibits GzmB activity PROGREFFE foundation grants, a Roche Organ Transplantation
(Ac-IEPD-CHO; BioVision, San Francisco, CA). All antibodies were Research Foundation and European Society for Organ Trans-
used at 10 mg/ml concentration. plantation grant, and under the auspices of the IHU-Cesti project,
which received French government financial support managed by the
Microarrays National Research Agency via the “Investment Into The Future”
Purified B cell samples from 32 individuals (10 TOLs, 10 HVs, and 12 program (ANR-10-IBHU-005). The IHU-Cesti project is also sup-
STAs) were analyzed with whole-genome Agilent human microarray ported by Nantes Metropole and the Pays de la Loire Region. This
as previously described24 according to the manufacturer’s instruc- work was also supported by the Labex IGO project (ANR-11-LABX-
tions (Agilent Technologies). Hybridization signals were normalized 0016-01) funded by the Investissements d’Avenir French Govern-
using a Lowess procedure.64 Probe conversion was performed using ment program, managed by the French National Research Agency.
MADGene65 and those pertaining to the same gene were averaged. The The research leading to these results has received funding from the
expression of 60 markers selected either for their participation in the European Union Seventh Framework Programme (FP7/2007–2013;
regulation of B cell functions26 and/or their implication in a tolerance- grant agreement 305147 BIO-DrIM).
related B signature27 was then investigated. Significance of differential
expression was evaluated using a one-tailed t test and results were com-
pared with those obtained on PBMCs.27
DISCLOSURES
None.
GzmB+ B Cell Phenotyping
Anti–human mAbs included the following: CD19-V450 CD38-FITC,
CD24-PE, CD5-APC, CD1d-PE, and GzmB-Alexa Fluor 700 from
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