Immunologic Human Renal Allograft Injury …jasn.asnjournals.org/content/25/7/1575.full.pdfImmunologic Human Renal Allograft Injury Associates with an Altered IL-10/TNF-a Expression

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Immunologic Human Renal Allograft Injury Associateswith an Altered IL-10/TNF-a Expression Ratio inRegulatory B Cells

Aravind Cherukuri,*† David M. Rothstein,‡ Brendan Clark,† Clive R. Carter,† Adam Davison,§

Maria Hernandez-Fuentes,| Eric Hewitt,¶ Alan D. Salama,** and Richard J. Baker*

*Renal Unit and †Department of Transplant Immunology, St. James’s University Hospital, Leeds, United Kingdom;‡Thomas E. Starzl Transplant Institute, Pittsburgh, Pennsylvania; |Nephrology and Transplantation, King’s CollegeLondon, London, United Kingdom; §Faculty of Medicine and Health and ¶Faculty of Biological Sciences, University ofLeeds, Leeds, United Kingdom; and **University College London Centre for Nephrology, Royal Free Hospital,London, United Kingdom

ABSTRACTHuman B cells with immunoregulatory properties in vitro (Bregs) have been defined by the expression ofIL-10 and are enriched in various B-cell subsets. However, proinflammatory cytokine expression in B-cellsubsets is largely unexplored.We examined the cytokine profiles of human PBMCs and found that subsetsof CD24hiCD38hi transitional B cells (TrBs), CD24hiCD27+ memory B cells, and naïve B cells express IL-10and the proinflammatory cytokine TNF-a simultaneously. TrBs had the highest IL-10/TNF-a ratio andsuppressed proinflammatory helper T cell 1 (Th1) cytokine expression by autologous T cells in vitromorepotently than memory B cells did, despite similar IL-10 expression. Whereas neutralization of IL-10 signif-icantly inhibited TrB-mediated suppression of autologous Th1 cytokine expression, blocking TNF-a in-creased the suppressive capacity of both memory and naïve B-cell subsets. Thus, the ratio of IL-10/TNF-aexpression, a measure of cytokine polarization, may be a better indicator of regulatory function than IL-10expression alone. Indeed, compared with TrB cells from patients with stable kidney graft function, TrBsfrompatients with graft rejection displayed similar IL-10 expression levels but increased TNF-a expression(i.e., reduced IL-10/TNF-a ratio), did not inhibit in vitro expression of Th1 cytokines by T cells, and abnor-mally suppressed expression of Th2 cytokines. In patientswith graft dysfunction, a low IL-10/TNF-a ratio inTrBs associatedwith poor graft outcomes after 3 years of follow-up. In summary, these results indicate thatB cell–mediated immune regulation is best characterized by the cytokine polarization profile, a finding thatwas confirmed in renal transplant patients.

J Am Soc Nephrol 25: 1575–1585, 2014. doi: 10.1681/ASN.2013080837

B lymphocytes have functions beyond the pro-duction of antibodies.1–9 An immunoregulatoryrole for B cells, first described in murine coli-tis,10 has since been supported by studies in avariety of autoimmune and alloimmune mod-els.11–16 Regulatory B-cell (Breg) activity is char-acteristically IL-10 dependent. Murine B cells alsoexpress a variety of proinflammatory cytokinesthat can polarize T cells in vitro and markedly in-fluence the immune response to pathogens invivo.5,9 However, the phenotype of B cells express-ing proinflammatory cytokines is largely un-known.

In humans, CD19+CD24hiCD38hi transitionalB cells (TrBs) isolated from peripheral blood wereshown to be enriched for IL-10 expression, andinhibit inflammatory cytokine expression by

Received August 6, 2013. Accepted December 18, 2013.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Aravind Cherukuri, Renal Unit, LincolnWing, St. James’s University Hospital, Beckett Street, Leeds, LS97TF, UK. Email: [email protected]

Copyright © 2014 by the American Society of Nephrology

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autologous CD4+ T cells in vitro.17 In patients with SLE, TrBswere defective in IL-10 expression and unable to suppresshelper T cell 1 (Th1) responses in vitro.17 By contrast, anotherstudy showed that human Bregs, defined by IL-10 expression,were enriched in the CD19+CD24hiCD27+ “memory” sub-population18 with increased B-cell IL-10 expression in pa-tients with autoimmune disorders.

Although IL-10 expression by putative regulatory B-cellsubsets has been studied, the concomitant expression ofproinflammatory cytokines, possibly by the very same B cells,and their effect on regulatory activity in vivo or in vitro is un-known. Such insights may aid further description of Breg sub-sets and clarify discrepancies in the literature regarding theinfluence of B cells in inflammatory settings.

In this study, we characterize B cells in human peripheralblood based upon both proinflammatory (TNF-a) and anti-inflammatory (IL-10) cytokine expression and show that asimple ratio of IL-10 to TNF-a best characterizes B-cell im-mune regulatory function. Based on the IL-10/TNF-a ratio,we show that TrBs exhibit the most anti-inflammatory cyto-kine profile in vitro. Whereas TrBs in renal allograft recipientswith stable function were similar to healthy controls, thosefrom patients with rejection were quantitatively and qualita-tively altered. Remarkably, a reduced TrB IL-10/TNF-a ratiowas predictive of worse clinical outcome.

RESULTS

Human (CD19+CD24hiCD38hi) TrBsExhibit the Most Polarized Anti-Inflammatory Cytokine Profile Basedon IL-10 and TNF-a ExpressionPBMCs from 15 healthy volunteers wereanalyzed and divided into three distinctB-cell (CD19+) subsets;CD272CD24hiCD38hi

TrBs, CD24hiCD27+ memory B cells, andCD272CD24+CD38+ naïve B cells (Figure1, A–C) as previously described.18,19 Fur-ther phenotypic characterization of bothnaïve and TrBs revealed that TrBs wereCD20hi, CD10+, and IgMhi, whereas naïveB cells were CD20+, CD102, and IgMint

(Figure 1D), consistent with previous re-ports.19,20

Next, magnetic bead–enriched (CD19+)B cells were stimulated with CpG andCD40L for 48 hours and analyzed for cyto-kine expression by intracellular staining, aspreviously described.18 Both memory andTrBs were enriched for IL-10 expressioncompared with naïve B cells17,18 (Figure2, A and B). These B-cell subsets also ex-pressed the proinflammatory cytokine,TNF-a, at a surprisingly high frequency,which approximated or even surpassedthat of IL-10 expression. TrBs contained

the fewest TNF-a+ B cells (Figure 2, C and D). Taken together,TrBs had the highest IL-10/TNF-a ratio, followed by memoryB cells, whereas naïve B cells had the lowest (Figure 2E). Thus,CD24hiCD38hi TrBs exhibit the most anti-inflammatory cyto-kine profile. To address the possibility that in vitro stimulationcould alter phenotype, B subsets were first purified and thencultured and cytokine expression was analyzed by ELISA.These results paralleled those obtained above, in which cellswere assessed for phenotype after in vitro culture (Figure 2F).

We next sought to determine whether individual B cellssimultaneously express more than one cytokine, using dual-intracellular staining. This analysis revealed that a significantfraction of B cells coexpressed both IL-10 and TNF-a. Al-though TrBs had the highest frequency of B cells expressingIL-10 alone, memory B cells had the largest fraction of dualpositive cells, and both memory and naïve B-cell populationswere enriched for cells expressing TNF-a+ alone (Figure 2, Gand H). The nature of cytokine polarization within theseB-cell subsets remained stable when analyzed at 12, 24, and48 hours (Figure 2I).

TrBs Selectively Inhibit Th1 Cytokine Expression in anIL-10–Dependent FashionTo determine the regulatory capacity of each B-cell subset,purified populations from healthy volunteers were examined

Figure 1. Characterization of transitional, memory, and naïve B subsets. (A–C) Definitionof human B subsets including CD19+CD24hiCD38hiCD272 TrB cells, CD19+CD24+CD38+

CD272 naïve B cells, and CD19+CD24hiCD27+ memory B cells. (D) Representative overlayhistograms comparing the strength of expression of CD20, CD10, and IgM between theTrB and mature naïve B cells (n=5).

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for their ability to inhibit cytokine expression by anti-CD3stimulated autologous CD4+CD252 conventional T cells(Tconvs). The regulatory activity of B-cell subpopulations

was compared with that of an equal num-ber of CD4+CD25hi regulatory T cells(Tregs) in the same assay (Figure 3). Sort-ing gates and purities of the cell subsets areshown in Supplemental Figure 1. TrBs weremore potent at selectively suppressingTNF-a and IFN-g expression by Tconvswith no significant effect on IL-4, com-pared with memory or naïve B cells, andsimilar in potency to Tregs (Figure 3).Thus, the strength of suppression in vitrocorrelates with and supports use of the IL-10/TNF-a ratio to define regulatory B cells(Bregs). The addition of neutralizing anti–IL-10 antibody to TrB cocultured with au-tologous Tconvs, restored both TNF-a(Figure 4, A and B) and IFN-g expression(Figure 4, C and D) by CD4 cells to thebaseline levels observed when Tconvswere cultured alone. Thus, regulatory ac-tivity of TrBs was IL-10 dependent. The in-fluence of TNF-a on regulatory function ofmemory or naïve B cells was analyzed byadding neutralizing antibodies to its recep-tors, TNFR1 and TNFR2, during coculturewith Tconvs (Figure 4). NeutralizingTNFR1 increased the ability of memoryB cells to suppress TNF-a and IFN-g ex-pression to levels comparable with thoseobtained with TrBs. Neutralization ofTNFR2 was somewhat less effective thanneutralizing TNFR1. Neutralizing TNFR1and TNFR2 on naïve B cells also increasedsuppression, but the effect was muchsmaller than the effect on memory B cells,perhaps as a result of lower IL-10 expres-sion by the naïve subset. In summary, neu-tralizing IL-10 inhibited the regulatoryeffects of TrBs, whereas blocking TNF re-ceptors significantly improved the suppres-sive capacity of both memory and naïveB cells. This highlights the significance ofboth IL-10 and TNF-a in regulation ofCD4 T cells by B-cell subsets. SupplementalFigure 2 shows the effect of TrB onCD45RA+ and CD45RA2 CD4 T cells.

Renal Transplant Recipients withRejection Exhibit Qualitative andQuantitative Differences in TrBsHaving established the regulatory proper-ties of TrBs, we investigated whether quan-

titative differences exist in renal transplant patients with stableallograft function versus rejection. TrBs from healthy volun-teers (n=15) were compared with 88 renal allograft recipients

Figure 2. Analysis of IL-10 and TNF-a expression by B subsets. Magnetic bead–enrichedCD19+ B cells are stimulated with CpG and CD40L for 48 hours plus phorbol ester,ionomycin, and brefeldin A (last 5 hours). (A and C) Each representative dot plot showsthe frequencies of IL-10+ or TNF-a+ B cells within the respective B subsets. (B and D)Cumulative frequency (mean6SEM) of IL-10 (*P,0.001, TrB versus memory or naive) andTNF-a producing cells (*#P=0.001, TrB versus memory; P=0.01, TrB versus naïve) withineach B cell subset in 15 healthy volunteers. (E) Analysis of the ratio of IL-10+/TNF-a+ cellswithin B cell subsets analyzed by flow cytometry (¥P=0.002, TrB versus memory;*P,0.001, TrB versus naïve). (F) Cumulative results expressed as mean6SEM for theIL-10/TNF-a ratio obtained from purified B cell subsets and analyzed by ELISA in 11healthy volunteers (*P,0.001, TrB versus memory or naive). (G) Representative dot plotfor the combined analysis of IL-10 and TNF-a by dual staining in the B subsets. (H)Cumulative results (mean6SEM) from six healthy volunteers of the distribution of eitherIL-10+ or TNF-a+ or mixed cytokine-positive B cells (*P,0.001; #P=0.01; **P=0.001;##P,0.001, in comparison with TrBs). (I) Analysis of IL-10+ and TNF-a+ B subsets at 12,24, and 48 hours in five independent controls after stimulation with CpG and CD40L. Thegraphs represent the IL-10/TNF-a ratio obtained for the respective B-cell subset at thethree different time points in five healthy volunteers. Statistical analysis is performed byANOVA using Tukey post hoc correction for multiple comparisons.

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(26–320months after transplantation)whowere predominantlynonsensitized recipients of first deceased donor transplants(79%) treated with basiliximab induction and maintained oncalcineurin inhibitors and mycophenolate mofetil withoutmaintenance steroids (Tables 1 and 2). Transplant recipientswith stable function (n=41) were compared with 47 patientswho underwent biopsies for graft dysfunction. On the basis ofgraft histology, this groupwas divided into recipients with graftdysfunction and no rejection (GD-NR; n=22) and recipientswith graft dysfunction with rejection (GD-R; n=25). The meantime to PBMC sampling from the date of the biopsy was15.869.3 days for the GD-NR group and 18.5617.9 days forthe GD-R group (P=NS). Clinical parameters for these partic-ipants are detailed in Table 2. Significant differences betweenthe groups included a longer time since transplantation for GDgroups and a higher usage of maintenance prednisolone asimmunosuppression in the GD-R group, in part reflecting achange made at the time of diagnosis for the six patients with aconcomitant diagnosis of T cell–mediated rejection and thenine patients whowere taking long-termmaintenance steroids.No other specific therapeutic interventions were initiated inthese patients before blood sampling. Histologic classification

of the GD-R group is detailed in Figure 5and graft dysfunction in this group waslargely attributable to chronic antibody-mediated rejection, although 25% of thepatients also had a component of T cell–mediated rejection. Eighty percent ofthese patients (20 of 25) had a donor-specific antibody detected at the time ofthe biopsy.

TrBs from patients with stable allograftfunctionwere similar to those from healthyvolunteers in terms of frequency (propor-tionof total B cells), absolute number, IL-10expression, and IL-10/TNF-a ratio (Figure6, A–D), although the mean value for eachparameter was slightly reduced (not statis-tically significant). Whereas patients withGD-NR had a lower absolute number ofTrBs compared with healthy volunteersand patients with stable allograft function,the trend toward a slightly lower IL-10/TNF-a ratio was not significant. By con-trast, patients with rejection (GD-R)showed a significant decrease in the per-centage and, importantly, in the IL-10/TNF-a ratio of TrBs compared with allother groups. The GD-R group had signif-icantly higher TNF-a expression(13.461.3 for patients with stable allograftfunction versus 21.661.9 for patients withGD-R; P,0.001) and a relatively lowerIL-10 expression (statistically not signifi-cant) that explains the significantly lower

IL-10/TNF-a ratio (Figure 6, A–D). Thus, transplant patientswith rejection display a quantitative decrease in Bregs and achange in their cytokine polarization, compared with thosewith stable graft function or GD-NR. Some patients withGD-R received corticosteroids for cellular rejection beforehaving their blood sampled for this study. However, whenonly patients with GD-R who did not receive steroids are con-sidered, they still have a significantly lower IL-10/TNF-a ratiocompared with either healthy volunteers or patients with sta-ble graft function (Supplemental Figure 3). Of note, despitethe decrease in IL-10/TNF-a ratio in TrBs in patients withGD-R, the IL-10/TNF-a ratio for the total B-cell populationis similar across all groups (Figure 6E).

To determine whether the observed difference in thecytokine profile of TrBs between patient groups correlateswith qualitative differences in regulatory function, we exam-ined the ability of TrB to inhibit cytokine expression byautologous Tconvs in vitro. Purified TrBs from transplant pa-tients with stable allograft function and those from healthyvolunteers exhibited similar inhibition of IFN-g and TNF-aexpression by Tconvs, while sparing IL-4 expression (Figure 6,F and G). By contrast, TrBs from patients with GD-R exerted a

Figure 3. TrB cells with a high IL-10/TNF-a ratio selectively suppress inflammatorycytokines by conventional T cells. Magnetic bead–enriched Tconv cells (CD4+CD252)are stimulated for 72 hours with plate-bound anti-human CD-3 (0.5 mg/ml) in thepresence of TrB (CD24hiCD38hi) B cells, CD24hiCD27+ memory B cells, CD24+CD38+

naïve B cells, or CD4+CD25hi Treg cells. Phorbol ester, ionomycin, and brefeldin A areadded for the last 6 hours of cell culture. Data are derived from six healthy volunteers.(A) Representative dot plots showing frequency of TNF-a, IFN-g, and IL-4 expressionamong Tconvs in the respective cell cultures. (B) Cumulative percentage stimulation/inhibition of the frequency of TNF-a+, IFN-g+, or IL-4+cells (mean6SEM) after culturewith Tregs or the respective B subsets. (*P,0.05, TrB versus CD24hiCD27+; **P=0.03,TrB versus CD24+CD38+; #P=0.02, TrB versus CD24hiCD27+; ##P=0.01, TrB versusCD24+CD38+).

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minimal effect on TNF-a and IFN-g expression by Tconvs,while significantly suppressing IL-4 expression. As a control,we examined Tregs from the same patients (Figure 6G). Unliketheir B-cell counterparts, Tregs from patients with rejectionwere no different in their activity than those from patientswith stable function or healthy controls. Because the samenumbers of TrBs were added in each assay, the principal dif-ference in TrBs from patients with GD-R and patients withstable allograft function was a decrease in their IL-10/TNF-a

ratio. These results confirm the advantageof the IL-10/TNF-a ratio over IL-10 ex-pression alone in predicting Breg activity.

Low IL-10/TNF-a Ratio Is Associatedwith Adverse Clinical OutcomesFinally, we analyzed the relationship betweencytokine polarization within the TrB subsetand graft outcomes. The IL-10/TNF-a ratiowas reasonably strong in distinguishing pa-tients with stable grafts from those with re-jection by receiver operation characteristiccurve analysis with an area under the curveof 0.823 (P,0.001) (Figure 7A). In patientswith graft dysfunction, when the rate of de-terioration of graft function was assessedover a 3-year follow-up from the time of thebiopsy (n=44), patients with an IL-10/TNF-aratio of TrBs below the group mean of 0.75(low ratio) had significantly more graft dete-rioration (measured by DeGFR) comparedwith patients with a ratio above the groupmean (high ratio) (Figure 7B). Moreover, alow IL-10/TNF-a ratio among TrBs was as-sociated with a worse outcome (defined byeither a doubling in serum creatinine fromthe time of the biopsy, or graft loss), with atrend toward poorer graft survival when thiswas analyzed for the GD-R group alone (Fig-ure 7, C and D). A similar, although nonsig-nificant, trendwith diverging curves was seeneven when the analysis was repeated withgraft loss as an end point (Supplemental Fig-ure 4). Thus, functional variation within theTrBs was clinically significant and was associ-ated with adverse graft outcomes.

DISCUSSION

Bregs have been shown toplay an importantrole in regulating immune responsivenessthrough IL-10 expression in a variety ofanimal models.10–12,14–16,21–25 Their role inhuman disease is less clear with studiesshowing that IL-10 is enriched in either

TrB (CD24hiCD38hi) or memory B cells (CD24hiCD27+) andIL-10 expression may be unchanged, elevated, or reduced inpatients with various autoimmune diseases.4,17,18,26,27

We demonstrate for the first time that Bregs in renaltransplant recipients with rejection are both reduced innumber and altered in function, compared with either healthycontrols or immunosuppressed patients with stable allograftfunction. By directly comparing human TrB and memoryB cells fromhealthy volunteers, which have been reported to be

Figure 4. The IL-10/TNF-a ratio predicts the capacity of the B subsets to suppress au-tologous Th1 cytokines. Magnetic bead-enriched Tconvs (CD4+CD252) are stimulated for72 hours with plate-bound anti-human CD-3 (0.5 mg/ml) in the presence of TrB(CD24hiCD38hi) B cells, CD24hiCD27+ memory B cells, CD24+CD38+ naïve B cells, orCD4+CD25hi Treg cells with either an isotype control or neutralizing antibodies to IL-10,TNFR1, TNFR2, or a combination of TNFR1 and TNFR2. Phorbol ester, ionomycin, andbrefeldin A are added for the last 6 hours of cell culture. Data are derived from threehealthy volunteers. (A and C) Representative scatter plots showing the detection of TNF-aand IFN-g by intracellular staining when Tconvs are cultured alone or in the presence ofTrB, memory, and naïve B cells with matched isotype controls or neutralizing antibodiesto IL-10, TNFR1, or TNFR2. Appropriate isotype controls (negative controls) for cytokinestaining labeled as “neg” are included for defining the cytokine positive gates for re-spective cell subsets. (B and D) Cumulative percentage stimulation/inhibition of the fre-quency of TNF-a+ ($P=0.01; *P=0.02; **P=0.10; #P=0.20; ##P=0.80) and IFN-g+ (©P=0.03;DP=0.01; @P=0.04; fP=0.2; �P=0.1) T cells (mean6SEM) after culture with the respectiveB subsets and neutralizing antibodies. Statistical analysis is performed by a paired t test.

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enriched for IL-10+ B cells,17,18 we have shown that the TrBsubset is most potent in suppressing proinflammatory cyto-kine expression by Tconvs in vitro. Importantly, this is not dueto a higher frequency of IL-10–expressing cells within the TrBpopulation; rather, increased activity is associated with dimin-ished expression of proinflammatory cytokines. Indeed,B cells within naïve, memory, and TrB subsets are not onlycapable of expressing IL-10, but also TNF-a and individualcells can express both cytokines concomitantly. Our data dem-onstrate that a high IL-10/TNF-a ratio is a better indicator ofpolarization toward anti-inflammatory cytokines and B-cellregulatory activity than IL-10 expression alone. This mayhelp explain inconsistencies between earlier studies.

The validity of the IL-10/TNF-a ratio as amarker for Bregs issupported by its ability to predict the potency of the B-cell sub-sets with equal IL-10 expression to suppress proinflammatorycytokine expression by Tconvs in vitro. Importantly, whereasneutralizing IL-10 blocked the suppressive activity of TrBs onautologous Th1 cytokine expression, neutralizing the effects ofTNF-a by blocking its receptors increased the in vitro suppres-sive capacity of both memory and naïve B cells. The value of theIL-10/TNF-a ratio is further substantiated by predicting sup-pressive activity of TrBs from renal transplant patients with dif-ferent clinical status. Despite exhibiting the same frequency ofIL-10 expression, TrBs from patients with GD-R displayed a sig-nificantly lower IL-10/TNF-a ratio than other groups, including

patients with graft dysfunction but without rejection (GD-NR).In addition, TrBs from rejecting patients were unable to inhibitTh1 cytokines while inhibiting Th2 cytokine production, in di-rect contrast with TrBs from transplant recipients with stablefunction. This suggests that aberrant immune regulation byBregs in the setting of allograft rejection and is consistent withmurine models in which Th1 responses are deleterious and Th2responses promote engraftment.28 Moreover, in patients withGD-R, the change in IL-10/TNF-a ratio was compounded by adecrease in the number of TrBs. Although causality cannot be es-tablished in this retrospective study, a reduced IL-10/TNF-a ratiowithin TrBs not only correlates with chronic antibody-mediatedrejection (CAMR), but a low ratio is associated with adverse graftoutcomes among patientswith graft dysfunction. This suggests thatthe IL-10/TNF-a ratio of TrB cells may be a useful supplement toexisting biomarkers of graft outcome, such as donor-specific anti-bodies, and warrants further prospective evaluation.29,30

There are several important consequences of this B cellfunctional heterogeneity. First, TrBs are not only morepolarized toward an anti-inflammatory function as a subset,but individual cells within that subset aremore likely to expressIL-10 alone (without TNF-a coexpression) compared witheither memory or naïve B cells. Thus, although the total num-ber of naive and especially memory B cells expressing IL-10 isnot substantially lower than TrBs, a greater proportion of theformer express or coexpress inflammatory cytokines and

Table 1. Patient characteristics

Clinical ParameterHealthy Volunteers

(n=15)Patients with Stable

Allograft Function (n=41)Patients withGD-NR (n=22)

Patients withGD-R (n=25)

P Value

Age, yr (95% CI) 45 (41 to 49) 49 (44 to 53) 51 (45 to 58) 49 (43 to 55) NSMen 61 66 64 64 NSWhite ethnicity 68 91 92 95 0.03a

Primary renal diagnosis NSGN N/A 15 45 28 0.01b

Hypertension/diabetes N/A 15 9 4Inherited N/A 15 14 16Othersc N/A 55 32 52

Transplantation detailsGraft number N/A 1.06 (1 to 1.15) 1.26 (1.04 to 1.48) 1.1 (0.95 to 1.26) NSType of allograft donor NSDonation after circulatory death N/A 12 14 12Donation after brain death N/A 68 68 64Live donor N/A 20 18 24

Donor cause of death NSTrauma N/A 15 29 20Vascular N/A 77 47 53Other N/A 8 24 27

HLA mismatches N/A 2.6 (2.1 to 3.1) 1.6 (1.0 to 2.3) 2.9 (2.5 to 3.5) 0.02d

Donor age, yr (95% CI) N/A 39 (33 to 45) 47 (38 to 55) 42 (35 to 49) NS

Data are presented as percentages ormedians (interquartile ranges) unless otherwise indicated. All categorical variableswere compared using the chi-squared test.All continuous variables are analyzed using ANOVA with Tukey post hoc test for multiple comparisons if normally distributed and the Kruskal–Wallis test forvariables with skewed distribution. Adjusted P values for multiple comparisons after the post hoc tests are presented. P,0.05 is significant. N/A, not applicable.aSignificant differences between healthy volunteers and the rest of the study population.bSignificant difference between patients with stable allograft function and the GD-NR group.cOthers include obstruction, chronic pyelonephritis, and unknown causes.dSignificant difference between GD-NR and other groups (stable allograft function versus GD-NR, P=0.02; GD-NR versus GD-R, P=0.01).

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appear less able to suppress T-cell responses. Moreover, indifferent clinical or pathologic states, the degree of polariza-tion can change. These observations cast doubt over the abilityof any phenotypic subset or single cytokine to adequately de-fine human Bregs. Rather, our findings indicate that cytokinepolarization is a better predictor of Breg activity.

Definitivemurine studies have shown that the expression ofproinflammatory cytokines by B cells can markedly affectimmune responses, for example by promoting Th1 differen-tiation or protective antibody responses to infectious patho-gens.5,8,31 It has been reported that B cells from patients withrelapsing multiple sclerosis express significantly more proin-flammatory cytokines than those from healthy controls, al-though cytokine expression was not actually correlated withdisease activity nor was the phenotype of such B cells exam-ined.32 However, B-cell depletion with rituximab reducedT-cell proliferation and expression of proinflammatory cyto-kines. Our study highlights the importance of identifying thebalance of proinflammatory and anti-inflammatory cytokineexpression within specific B-cell subsets.

Recent studies showed that renal transplant patients whowere “operationally tolerant” exhibit higher numbers of TrBsand IL-10 expression than nontolerant patients with stablerenal function.33,34 In this study, patients with stable allograftfunction (on immunosuppression) have a similar number ofand IL-10 expression by TrBs as healthy volunteers. Moreover,we recently showed that stable renal transplant patients withsuperior graft function, irrespective of immunosuppressiveregimen, have higher numbers of both total B cells and TrBscompared with those with reduced renal function.35 In thisstudy, not only are TrBs reduced in the setting of rejection, buttheir cytokine profile and functional activity upon in vitrononcognate polyclonal stimulation are abnormal. Closer in-spection reveals that most of the patients (80%) in the GD-Rgroup exhibited microvascular inflammation and scarringwith or without complement (C4d) deposition, and serologicevidence of circulating donor-specific HLA antibody—all fea-tures of CAMR. In 25% of these patients, a component ofacute cellular rejection was superimposed. Whether Breg ab-normalities precede the development of antibody-mediated or

Table 2. Allograft characteristics at the time of biopsy

Clinical Parameter Healthy VolunteersPatients with StableAllograft Function

Patientswith GD-NR

Patientswith GD-R

P Value

Allograft functionAge of the graft (mo)a N/A 56 (43 to 78) 78 (61 to 128) 83 (51 to 153) 0.04b

Best eGFR achieved N/A 74 (69 to 79) 59 (48 to 69) 66 (56 to 76) 0.01c

eGFR (ml/min) N/A 65 (61 to 70) 31 (25 to 36) 29 (22 to 36) ,0.001d

Proteinuria (g/L) N/A 0.17 (0.13 to 0.20) 0.84 (0.51 to 1.18) 1.47 (0.67 to 2.26) 0.01d

Maintenance immunosuppressionCNI N/A 97 92 83 NSMMF/azathioprine N/A 71 76 78 NSPrednisolone N/A 7 18 60 0.01e

DSA-HLA class-1% positive N/A 9 2.5 48 ,0.001e

DSA-HLA class-2% positive n/a 0 2.5 36 ,0.001e

Late cellular rejection n/a 0 23 25Allograft pathology N/A N/ARecurrent GN N/A N/A 27Othersf N/A N/A 32IFTA (scarring) N/A N/A 23Viral infection 9CNI toxicity N/A N/A 9

Chronic antibody-mediated changesGlomerulitis N/A N/A 0 44Transplant glomerulopathyg N/A N/A 0 64Peritubular capillaritis N/A N/A 0 36Peritubular capillary C4d deposition N/A N/A 0 48

Data are presented as percentages. All categorical variables were compared using the chi-squared test. All continuous variables are analyzed using ANOVA withthe Tukey post hoc test for multiple comparisons if normally distributed and the Kruskal–Wallis test for variables with skewed distribution. Adjusted P values formultiple comparisons after thepost hoc tests are presented. P,0.05 is significant. N/A, not applicable; CNI, calcineurin inhibitor (cyclosporine or tacrolimus); MMF,mycophenolate mofetil/mycophenolate sodium; DSA, donor-specific antibody; IFTA, interstitial fibrosis and tubular atrophy (no obvious cause identified).aBecause graft age (vintage) is extremely skewed, median and interquartile ranges have been presented and data were analyzed by a nonparametric test.bSignificant difference between stable allograft function and GD-R.cSignificant difference between stable allograft function and GD-NR.dSignificant difference between stable allograft function and GD-NR; stable allograft function and GD-R.eSignificant differences between GD-R and the rest of the study population.fOther pathology includes urinary tract obstruction or vascular problems (two patients with definitive evidence of obstructive uropathy and one patient with renalartery stenosis were not biopsied).gChronic transplant glomerulopathy with basement membrane multilayering.

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cell-mediated rejection (suggesting causality) or occur as a re-sult of these processes must await prospective longitudinalstudy. This is important because CAMR is a major cause ofchronic renal allograft loss.36–38

One limitationof this study is that sixpatientswhoexhibitedan acute cellular component to their rejection on biopsies wereinitiated on treatment with corticosteroids before bloodsampling. Thus, more patients with rejection were on main-tenance corticosteroids. However, as noted above, patientswith GD-R who did not receive corticosteroids still had asignificantly lower IL-10/TNF-a ratio than other patientgroups. Finally, it should be noted that the GD-NR groupwas heterogeneous and some patients had “immune-mediated”pathology such as recurrent GN. This may explain why thisgroup exhibited some reduction in TrBs and IL-10/TNF-a ratio(not significant) compared with patients with stable allograftfunction and healthy volunteers, while remaining significantlyhigher than the GD-R group. In this study, although the in vitrosuppressor assay utilized nonantigen-specific stimulation ofT cells, it correlated well with IL-10/TNF-a ratio and clinicaldiagnosis and was predictive of 3-year outcomes. Validation ofAg-specific T- and B-cell responses in this in vitro assay willrequire further study.

Our study provides novel insights into the description ofB cell–mediated immunoregulation in humans. Because a sig-nificant number of B cells, even in IL-10–enriched subsets, canexpress proinflammatory cytokines that can bemodulated in thecourse of immune-mediated disease, we contend that any defini-tion of the Breg phenotype should take this finding into account.Using such a strategy, we have shown that CD24hiCD38hi TrBsoutperformed other B-cell subsets in their regulatory po-tential, and their cytokine polarization profile correlated

with in vitro function and clinical status and was associatedwith clinical outcomes after biopsy in renal transplant re-cipients.

CONCISE METHODS

Study ParticipantsVenous blood samples were collected from healthy volunteers (n=15)

and renal transplant recipients (n=88) and PBMCs were isolated ac-

cording to standard criteria by Ficoll-Paque (Cedar Lane, Burlington,

ON, Canada) density gradient centrifugation. Written informed con-

sent was obtained from all of the participants according to the Decla-

ration of Helsinki. The study was approved by the York Regional Ethics

Committee (reference no. 08/H1311/41). Serum samples from the

transplant recipients were stored for the analysis of HLA-specific anti-

bodies. All renal transplant recipients followed up in our out-patient

clinic were screened for graft dysfunction based on serial serum creat-

inine and proteinuria assessments. Patients with proteinuria of.0.5g/

d assessed by urine protein creatinine ratio on three consecutive random

urine samples and/or deteriorating function as assessed by change in 1/

creatinine over a year39 were offered a transplant biopsy. On the basis of

the histology, they were grouped into patients with a histologic pheno-

type of rejection (GD-R, n=25) or patients with no evidence of rejection

(GD-NR, n=22). This group was randomly selected based on the time

elapsed since the date of the biopsy. Blood samples were collected over a

period of 18months. Patients who had a biopsy to blood sampling time

of.3 months were not included in the study. The details of the Banff

classification for the patients within the GD-R group are summarized in

Figure 3. Blood samples from these patients were collected for analysis

within 3months of the biopsywhen the patients attended for their clinic

appointments. A randomgroup of 41 patients with stable graft function

defined by stable creatinine and absence of proteinuria were used for

comparative analysis. This group of patients was not biopsied in light of

their stable renal function. The GD-R group was created on the basis of

graft dysfunction along with that of the presence of at least one of either

glomerulitis, peritubular capillaritis, and transplant glomerulopathy

with or without peritubular C4d deposition in the peritubular capillar-

ies in the presence of interstitialfibrosis and tubular atrophy.40 All trans-

plant recipients irrespective of their graft function were tested for the

presence of HLA-specific antibodies. Table 1 summarizes the character-

istics of patients and controls. None of the patients were commenced on

any other therapy for rejection before blood sampling apart from the

small subgroup of patients who received corticosteroids for the diagno-

sis of T cell–mediated rejection.

Antibodies, Flow Cytometry, Lymphocyte Isolation,and Cell SortingAll experiments were performed on fresh cells on the same day of blood

sampling. Anti-human mAbs included FITC-CD24 (ML5), PE-CD27

(M-T271), APC-CD27 (M-T271), PerCP-Cy5.5-CD38 (HIT2), PE-

CD1d (UCHT2), FITC-CD5 (UCHT2), APC-H7-CD19 (SJ25C1),

FITC-IgM (G20-127), PE-CD25(M-A251), PE-IL-10 (JES3-9D7), PE-

TNF-a (MAb11), AlexaFluor 488-TNF-a (MAb11), PE-IL4 (8D4-8),

AlexaFluor 488-IFN-g (B27), functional grade CD3 (HIT3a), and

Figure 5. Details of the Banff classification of the biopsies for pa-tients in the GD-R group. Classification is as follows: 0, absent; 1,mild; and .1, moderate to severe. These findings show that thebiopsies have a significant degree of chronic damage as suggestedby ct, cv, and ci scores. aah, arteriolar hyalinosis; c4d, C4d de-position in the peritubular capillaries; cg, chronic transplant glo-merulopathy with basement membrane multilayering; ci, interstitialfibrosis; ct, tubular atrophy; cv, fibrointimal thickening; g, glomerulitis;i, interstitial inflammation; mm, mesangial expansion; ptc, peritubularcapillaritis; t, tubulitis; ti, total inflammation; v, endothelitis.

1582 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1575–1585, 2014

CLINICAL RESEARCH www.jasn.org

neutralizing anti–IL-10 (JES3-19F1) from Beckton Dickinson. APC-

CD4 (OKT4), PerCP-Cy5.5-CD20 (2H7), PE-CD24 (eBioSN3),

APC-CD38 (HIT2), FITC-CD27 (LG.7F9), and PerCP-Cy5.5-

TNF-a (MAb-11) were from eBioscience (San Diego, CA). Neutral-

izing TNFR1 (clone 16805) and TNFR2 (clone 22210) antibodies

along with the isotype controls were from R&D Systems. Brilliant

violet 421-CD24 (M1/69) and AlexaFluor 700-CD27 (O323) were

from Biolegend.

Absolute lymphocyte numbers were calculated using BD-Trucount

beads asper themanufacturer’s instructions.Redcellswere lysedusingBD-

FACSlyse. Nonspecific FcR binding was blocked by 20% human

normal serum. Cursors were set using isotype and fluorochrome

matched negative controls for both surface and intracellular stain-

ing. Cells were then analyzed using a BD FACSCalibur flow cytom-

eter (Beckton Dickinson).

In some experiments, B cellswere isolated fromPBMCsby positive

selectionusingCD19magnetic beads (MiltenyiBiotec). In this setting,

B-cell purity was reexamined using anti-CD20. CD4+CD252 and

CD4+CD25hi T cells were also enriched using MACS magnetic bead

kits (Miltenyi Biotec). Various B-cell subsets were enriched by a

Moflo cell sorter (Beckman Coulter) based on their surface expres-

sion of CD19, CD24, CD38, and CD27.

Cell Culture-Cytokine DetectionMagnetic bead-enriched B cells were suspended in RPMI 1640 (Life

Sciences) supplemented with 10% FBS and 1% penicillin-streptomycin

L-glutamine (Sigma-Aldrich) at 13106 cells/ml and stimulated with

10 mg/ml CpG (CpG-ODN2006; Invivogen, San Diego, CA) and

1 mg/ml CD40L (Sigma-Aldrich). Next, 50 ng/ml PMA (Sigma-Aldrich)

and1mg/ml ionomycin (Sigma-Aldrich) and1ml/mlBrefeldinA(Beckton

Dickinson) were added for the last 5 hours of cell culture. Cultured cells

were stained with surface markers, fixed, and permeabilized using the

Cytofix/Cytoperm kit (BD Biosciences) and then stained for intracel-

lular cytokines. Alternatively, B-cell subsets were sort-purified and stim-

ulated with anti-CD40 and CpG for 48 hours, and then cytokines were

analyzed from the cell culture supernatants by bead-based ELISA (In-

vitrogen) using the manufacturer’s instructions. Because all experi-

ments were conducted on fresh PBMCs within a few hours of blood

sampling, the cell viability tested by trypan blue method was approxi-

mately 80%–85% even after 48 or 72 hours of cell culture.

Figure 6. CD19+CD24hiCD38hi TrB cells are altered qualitatively and quantitatively in renal allograft rejection. Bar graphs (mean6SEM)comparing healthy volunteers and patient groups for the percentage (A) and absolute number (B) of CD24hiCD38hi TrB cells (#P=0.03,S versus GD-R; *P=0.05, GDR versus GD-NR; **P,0.001, S versus GD-NR; ##P,0.001, S versus GD-R). (C–E) The percentage of IL-10 forTrBs, IL-10/TNF-a ratio for TrBs, and whole B cells (¥P,0.001, S versus GD-R; ɸP=0.03, GD-NR versus GD-R). (F) Representative dot plots forone of five patients in the GD-R group for the TNF-a, IFN-g, and IL-4 expression by Tconvs cultured alone or in the presence of TrBs orTregs. Numbers in each plot indicate the frequency of Tconvs expressing each cytokine under respective culture conditions. (G) Graphicalrepresentation (mean6SEM) of the cumulative results for the suppressive/stimulatory effect of the TrB cells or Tregs on the Tconv cytokineexpression in healthy volunteers (n=6), stable (n=7), and GD-R (n=5) groups ($P,0.001, S versus GD-R: TNF-a; $$P=0.002, IFN-g;$$$P,0.001, IL-4,) Statistical analysis is performed by ANOVA using Tukey post hoc correction. GD-NR, patients with graft dysfunction-norejection; GD-R, patients with graft dysfunction-rejection; HV, healthy volunteers; S, patients with stable graft function.

J Am Soc Nephrol 25: 1575–1585, 2014 Human Bregs and Immunologic Renal Allograft Injury 1583

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B Functional AssaysCell cultures of 13105 magnetic bead–enriched CD4+CD252

T cells and FACS-sorted CD24hiCD38hi or CD24hiCD27+ or

CD24+CD38+ B-cell subsets (1:1) were stimulated with 0.5 mg/ml of

plate-bound anti-human CD3 in 96-well u-bottom tissue culture

plates (Nunc). Brefeldin-A was added for the last 6 hours along with

PMA and ionomycin. Cells were surface stained with CD4-APC and

CD20-PerCp-Cy5.5 followed by fixation and permeabilization and

staining for intracellular cytokines. Cultures of CD4+CD252 cells

with CD4+CD25hi Tregs (1:1) were used as positive controls. To test

the utility of either IL-10 or TNF-a, neutralizing antibodies to IL-10 or

TNFR1 and TNR2 or isotype controls were added to the respective

wells of the culture plate.

Statistical AnalysesStatistical analysis was performed by SPSS (version 20) andGraphPad

Prism (version 6) for Windows. Categorical variables were compared

using the chi-squared test and continuous var-

iables using the paired t test. Analyses with mul-

tiple comparisons were completed using

ANOVAwith Tukey post hoc analysis to account

for the multiple comparisons in the case of

normally distributed variables or the Kruskal–

Wallis test for variables with a skewed distribu-

tion. Analysis of allograft outcomes over a

follow-up period was by the Kaplan–Meier

method and the groups were compared by the

log-rank test. A P value of,0.05 was considered

significant.

ACKNOWLEDGMENTS

This study was supported by educational grants

from the Yorkshire Kidney Research Fund and

the Leeds Teaching Hospitals Charitable Foun-

dation. The funders had no role in the study

design, data collection and analysis, decision to

publish, or preparation of the manuscript.

DISCLOSURESNone.

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See related editorial, “B Cells and Kidney Transplantation: Beyond Antibodies,”on pages 1373–1374.

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2013080837/-/DCSupplemental.

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