Calcineurin inhibitors dampen humoral immunity by acting directly on naive B cells

Calcineurin inhibitors dampen humoral immunity by acting directly on naive B

cells

Short title: In vitro effect of calcineurin inhibitors on humoral immunity.

Ruth De Bruyne 1, Delfien Bogaert

2,5, Natalie De Ruyck

3, Bart N. Lambrecht

4,

Myriam Van Winckel 1, Philippe Gevaert

3 and Melissa Dullaers

5

1 Department of Pediatric Gastroenterology, Hepatology and Nutrition, Princess

Elisabeth Children’s Hospital, Ghent University Hospital, Ghent, Belgium

2 Department of Pediatrics, Princess Elisabeth Children’s Hospital, Ghent University

Hospital, Ghent, Belgium

3 Upper Airways Research Laboratory, Department of Otorhinolaryngology, Ghent

University Hospital, Ghent, Belgium

4 Laboratory of Immunoregulation, VIB Inflammation Research Center, Ghent,

Belgium; Clinical Immunology Research lab, Department of Respiratory Medicine,

Ghent University Hospital, Ghent, Belgium; Department of Pulmonary Medicine,

Erasmus MC, Rotterdam, The Netherlands

5 Clinical Immunology Research Laboratory, Department of Respiratory Medicine,

Ghent University Hospital, Ghent, Belgium

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an‘Accepted Article’, doi: 10.1002/cei.12604

This article is protected by copyright. All rights reserved.

2

Corresponding author

Ruth De Bruyne

Department of Pediatric Gastroenterology, Hepatology and Nutrition,

Princess Elisabeth Children’s Hospital,

Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium

Phone number: +32 9 332 39 66; Fax number: +32 9 332 21 70

E-mail: [email protected]

Key words

B cells

Calcineurin inhibitors

Immunoglobulins

Immunosuppression

T follicular helper cells

Page 31 of 60 Clinical Experimental Immunology


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Abbreviations

BAFF; B cell activating factor

CNI; calcineurin inhibitors

CyA; cyclosporine A

FK506; tacrolimus

Ig; immunoglobulin

IL; interleukin

INF-γ; interferon-γ

IS; immunosuppression

PBMC; peripheral blood mononuclear cells

Tfh; T follicular helper cells

TNF; tumor necrosis factor

2 of 60Clinical Experimental Immunology


4

Abstract

Background

Calcineurin inhibitors (CNI), used frequently in solid organ transplant patients are

known to inhibit T cell proliferation but their effect on humoral immunity is far less

studied.

Methods

Total and naive B cells from healthy adult donors, were cultured in IgA- or IgG / IgE-

promoting conditions with increasing doses of cyclosporine, tacrolimus, rapamycin or

methylprednisolone. The effect on cell number, cell division, plasmablast

differentiation and class-switching was tested. To examine the effect on T follicular

helper (Tfh) cell differentiation, naive CD4+ T cells were cultured with IL-12 and

titrated immunosuppressive drug (IS) concentrations.

Results

Total B cell function was not affected by CNI. However, naive B cell proliferation

was inhibited by cyclosporine and both CNI decreased plasmablast differentiation.

Both CNI suppressed IgA, whereas only cyclosporine inhibited IgE class-switching.

Rapamycin had a strong inhibitory effect on B cell function. Strikingly,

methylprednisolone, increased plasmablast differentiation and IgE class-switching

from naive B cells. Differentiation of Tfh cells decreased with increasing IS doses.

Conclusion

CNI did affect humoral immunity directly by suppressing naive B cells. CNI, as well

as rapamycin and methylprednisolone, inhibited the in vitro differentiation of Tfh

from naive CD4+ T cells. In view of its potent suppressive effect on B cell function

and Tfh cell differentiation, rapamycin might be an interesting candidate in the

management of B cell mediated complications post solid organ transplantation.



5

Introduction

Significant progress has been made in preventing acute allograft rejection following

solid organ transplantation resulting in improved allograft survival. Chronic rejection,

however, remains a major hurdle menacing the long-term function of the transplanted

organs. Allo-immune responses primarily defined by the development of antibodies to

donor mismatched major histocompatibility antigens as well as the development of

auto-antibodies to organ specific self-antigens are involved in the pathogenesis of

chronic allograft rejection 1.

Furthermore, auto-immune disorders such as de novo autoimmune hepatitis post liver

transplantation 2 as well as de novo acquired allergic conditions post transplant are

well know complications after solid organ transplantation. The use of tacrolimus has

been associated with allergic diseases and elevated IgE in several transplant

populations 3-5

.

Since many years, calcineurin inhibitors (CNI), such as cyclosporine (CyA) and

tacrolimus (FK506), form the keystone of most maintenance immunosuppression

protocols in solid organ transplantation 6. Nowadays, the use of FK506 is preferred to

CyA because of its superiority in prevention and treatment of rejection 7,8

. CNI are

known to inhibit T cell proliferation by holding in nuclear factor of activated T cells

and interleukin (IL)-2 gene-transcription 9. Their effect on the humoral immune

response, however, is far less studied and remains to be elucidated 10

.

CNI are known to alter the balance between different CD4+ T cell subsets,

particularly by suppression of regulatory T cells 11-12

. More recently, an additional T

cell subset, known as T follicular helper (Tfh) cells, has been identified which are

CD4+

T helper cells specialized in providing help to B cells and regulating humoral

immune responses. Tfh cells control the development of antigen-specific B cell

Page 34 of 60Clinical Experimental Immunology


6

immunity 13

and have been implicated in autoimmune diseases 14-16

. Tfh cells are

essential for the formation and maintenance of germinal centers and for the generation

of most memory B cells and plasma cells 17,18

, providing CD40 ligand and multiple

cytokines, such as IL-2, IL-4, IL-10 and IL-21 19-22

. These signals promote B cell

proliferation, class switch recombination, and somatic hypermutation, resulting in

highly specific, class-switched plasma cells and long-lived memory B cells 23-25

. The

original feature used to identify Tfh cells is expression of chemokine receptor CXCR5

19,26. Another important characteristic is secretion of IL-21, which is the “signature”

cytokine of Tfh cells 17

. Immunosuppressive drugs, such as tacrolimus, could

influence humoral immunity by acting on B cells directly or on Tfh cells. Antigen,

CD40 and IL-21 are absolute requirements for T cell-dependent B cell activation

resulting in proliferation, class-switching and differentiation into memory B cells or

plasma cells. IL-10 and TGF-β1 drive IgA class-switching 25,27

, whereas IL-4 induces

IgE switching 28

.

Since very little data are available on the effect of immunosuppressive drugs on

humoral immunity, we examined the direct in vitro effect of immunosuppression

(FK506, CyA, rapamycin, methylprednisolone) on differentiation and class-switching

of total and naive B cells. Furthermore, we studied the effect of these

immunosuppressive drugs on Tfh cell differentiation from naive CD4+ T cells, as they

too influence the humoral immune response.

We here demonstrate that CNI do exert a direct effect on humoral immunity by

suppressing naive B cells. Tfh cell differentiation from naive CD4+ T cells was

inhibited by all immunosuppressive drugs used.

Materials and Methods



7

Cells and cell cultures

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque density

gradient (Miltenyi Biotec) from buffycoats of healthy adult blood donors (Blood

Transfusion Center Oost-Vlaanderen, Red Cross). The study was approved by the

ethics committee of Ghent University Hospital (BTC20130116).

B cells were isolated from the lymphocyte-rich fractions using Human B Cell

Enrichment Set (BD Biosciences, CA). To obtain naive B cells, the cells were stained

for CD3, CD19, IgD and CD27. CD27- IgD

+ naive B cells were sorted on a FACSAria

(BD). The purity of sorted B cells was generally >98%.

Total and naive B cells were cultured in 96-well U bottom plates with RPMI 1640

(Gibco) supplemented with 10% FCS, 100 U/ml penicillin (Sigma), 100 µg/mL

streptomycin (Sigma), 1% non-essential amino-acids (MEM NEAA, Gibco), 1 mM

sodiumpyruvate (Gibco) and 0,05 mM βmercapto-ethanol (Gibco) at 5 - 10 x 104

cells/200 µL/well in the presence of IL-2 (20 ng/ml; R&D Systems), anti-CD40

antibody (1 µg/mL, clone 12E12, kind gift of Sandra Zurawski, Baylor Institute,

Dallas, TX) and IL-21 (20 ng/mL; Invitrogen). Prior to culture, B cells were labelled

with 0,5 µM CFSE (Invitrogen) to track proliferation. Class-switching was induced

by adding respectively human recombinant TGF-β1 (1 ng/mL; R&D Systems) and

CpG (50 nM; ODN2006, InVivogen) for IgA class-switching and IL-4 (20 ng/mL;

R&D Systems) for IgG and IgE class-switching. Immunosuppressive drugs were

added, titrated in 3 different concentrations (final concentrations in culture):

methylprednisolone and FK506 (10 -9

M, 10 -8

M, 10 -7

M), rapamycin (0,1 ng/mL, 1

ng/mL, 10 ng/mL) and CyA (10 ng/mL, 100 ng/mL, 1000 ng/mL). The

immunosuppressive drugs were diluted in tissue culture medium (RPMI + additives)

at least 1/1000 for cyclosporine and at least 1/10 000 for methylprednisolone and



8

tacrolimus, the initial stock solutions were stored in ethanol. The medium

concentrations of FK506 (8,04 ng/mL) and CyA (100 ng/mL) are similar to target

through serum levels during the first year after liver transplantation. Target through

serum levels for rapamycin during the first year post transplant situate between the

medium and highest dose of rapamycin used in the experiments. Cells were incubated

at 37 °C. After 7 days, supernatant was harvested and cells stained for flow

cytometry.

Naive CD4+ T cells were isolated from PBMC of healthy adult donors by negative

selection (Human Naive CD4+ T Cell Isolation Kit II; Miltenyi Biotec). Cell purity

checked with flow cytometry was always ≥ 95%. Cells were cultured in 96-well U

bottom plates with complemented RPMI 1640 at 2 x 105 cells/200 µL/well in the

presence of IL-12 (20 ng/mL; R&D Systems), plate-bound anti-CD3 (5 µg/mL;

Biolegend), soluble anti-CD28 (2 µg/mL; Biolegend) to facilitate T cell

differentiation and induce Tfh cells 29

. Prior to culture, T cells were labelled with 0,5

µM Cell Trace Violet (CTV) (Invitrogen) to track proliferation. Titrated doses of

methylprednisolone, FK506, rapamycin and CyA were added (cfr supra). Cells were

incubated at 37 °C. Supernatant was harvested after 48 hours and cells stained for

flow cytometry.

Flow cytometry

Cells were stained with fixable viability dye 506 (eBioscience) and fluorescently

labelled monoclonal antibodies using manufacturers’ instructions. Following

monoclonal antibodies were used: anti-CD3, anti-CD19, anti-CD27, anti-CD38, anti-

human IgD, anti-human IgG, anti-human IgE (all BD Biosciences); anti-human IgA

(Miltenyi Biotech); avidin/streptavidin (eBioscience); anti-human CD4 (BioLegend).



9

To activate naive CD4+ T cells and subsequently measure their cytokine production

by intracellular flow staining, the cells were incubated with 100 µg/mL phorbol

myristate acetate (PMA, Sigma-Aldrich), 10 µg/mL Brefeldin A (Sigma-Aldrich) and

2 mM GolgiStop (BD Biosciences) for 5 hr at 37° C. Intracellular staining of CD4+ T

cells was performed using PermFix and PermWash (BD Biosciences) and anti-human

IL-21 (eBioscience) and interferon-γ (IFN-γ) (BioLegend).

Cells were sorted on a FACSAria (BD Biosciences) and acquired on a LSRFortessa

(BD Biosciences). At least 100 000 events per sample were recorded. Counting beads

(Flow Cytometry Absolute Count Standard, Full spectrum, Bang Laboratories) were

used to calculate the absolute number of cells based on the obtained cell percentages.

Data were analysed using FlowJo software (Tree Star, version 9.4.11).

ELISAs

ELISAs were performed to measure total IgG, IgE and IgA in the culture supernatant

(Human IgG, IgE and IgA ELISA Mabtech) according to the manufacturer’s protocol.

IFN-γ and IL-21 levels were measured in culture supernatant of naive CD4+ T cells

(Specific Human ELISA ‘Ready-SET-Go!’ 2nd

generation kits; eBioscience)

according to the manufacturer’s protocol.

Statistical Analysis

Data were analyzed using GraphPad Prism software. Differences between groups

were compared using Friedman test. Dunn’s multiple comparison test was performed

and adjusted p values given. A p value ≤ 0,05 was considered statistically significant.

To compensate for inter-donor variability, we normalized the data by dividing the

measured values by the value of the condition without immunosuppression of the

corresponding donor.



10

Results

As the direct effect of CNI on the humoral immune response is not well known, we

used an in vitro B cell class-switching system to test the effect of CNI, rapamycin and

methylprednisolone on several aspects of B cell differentiation.

Supplementary figure 1 illustrates the levels of IgA, E and G secretion obtained from

naive B cells with our in vitro class-switching system. IgA class-switching was

induced by IL-21+TGF-β1, both IgE and IgG class-switching were induced by IL-

21+IL-4 (in addition to anti-CD40 antibody and IL-2).

Effect of immunosuppression on number of cells, cell division and plasmablast

differentiation

Total and naive B cells were cultured in IgA- (TGF-β) or IgG / IgE- (IL-4) promoting

conditions in the presence of increasing doses of immunosuppression.

Both CyA and FK506 decreased the number of CD19+ B cells in the TGF-β condition

of total (p<0,001; p=0,002) and naive (p=0,002; p=0,02) B cells (figure 1, a-b). In

naive B cells, CyA had a strong dose-dependent inhibitory effect on division capacity

(p=0,02) whilst FK506 only suppressed cell division in the TGF-β condition of naive

B cells (p<0,001) (figure 1, c-d). CyA (p=0,02) and FK506 (p=0,02; p<0,001) both

significantly suppressed CD38+ plasmablast differentiation in naive but not in total B

cells (figure 1, e-f).

Rapamycin strongly suppressed B cell numbers in total B cells (p≤0,001) and in the

TGF-β condition of naive B cells (p=0,002) (figure 1, a-b). There was a strong dose-

dependent inhibitory effect on division capacity and CD38+ plasmablast

differentiation in total and naive B cells (all p<0,001) (figure 1, c-f).



11

In total B cells, methylprednisolone significantly increased B cell number in the IL-4

condition (p≤0,001) with the same trend present in the naive B cells (figure 1, a-b).

Strikingly, in naive B cells, methylprednisolone had a stimulating effect on CD38+

plasmablast differentiation in low and especially medium (p=0,007; p=0,001) doses.

In the medium concentration of the IL-4 condition, CD38+ plasmablasts nearly

doubled compared to the control condition without immunosuppression. This effect

was absent in total B cells (figure 1, e-f).

Effect of immunosuppression on Ig class-switching

In total B cells, CyA and FK506 did not exhibit any significant effect on either IgA or

IgE class-switching whilst both had a dose-dependent suppressive effect on IgA class-

switching (p=0,002; p<0,001) and IgA secretion (p=0,04; p=0,02) in naive B cells.

Moreover, increasing doses of CyA suppressed IgE class-switching (p=0,02) and IgE

secretion (p=0,04) in naive B cells but FK506 did not significantly affect IgE class-

switching or IgE secretion in these cells (figure 2, a-d).

Rapamycin inhibited IgA as well as IgE class-switching in total and naive B cells (all

p<0,001) (figure 2, a-b). Rapamycin also induced a significant dose-dependent

decrease in IgA secretion in total (p<0,001) and naive (p=0,02) B cells. IgE secretion

was suppressed in naive B cells only (p=0,001) (figure 2, c-d).

Methylprednisolone inhibited IgA (p=0,002) and IgE (p=0,02) class-switching in total

B cells (p≤0,02) (figure 2, a). In naive B cells, IgA class-switching was suppressed

(p=0,002) but IgE class-switching was induced in low and medium (p=0,003)

concentrations (figure 2, b). IgE secretion, on the contrary, was significantly

suppressed in naive B cells (p=0,02) (figure 2, d).



12

Rapamycin strongly decreased IgG secretion in all conditions of total (p<0,001) and

naive (p=0,01) B cells. There was no significant effect of CNI or methylprednisolone

on IgG secretion (figure 2, c-d).

CNI strongly suppress in vitro induction of IL-21 expressing Tfh cells

To examine the effect of immunosuppressive drugs on Tfh cell differentiation, we

cultured naive CD4+ T cells from healthy adult donors with IL-12 (to stimulate Tfh

cell skewing) 29

and titrated concentrations of CyA, FK506, methylprednisolone or

rapamycin.

The division capacity of CD4+ T cells was significantly suppressed by CyA, FK506,

rapamycin and methylprednisolone (p<0,001) (figure 3, b).

The frequency of IFN-γ+ (all p<0,001) and IL-21

+ (p<0,001 for CyA and FK; p=0,003

for methylprednisolone and rapamycin) single positive, as well as IFN-γ+

IL-21+

double positive (all p<0,001) CD3+

CD4+ T cells decreased under all four

immunosuppressive drugs although to a different extent, with CyA and FK506

exerting the strongest effect (figure 3, a). These results matched the results of the

cytokine secretion in culture supernatants (secreted INF-γ, all p<0,001; secreted IL-

21, p<0,001; p=0,002; p=0,03; p=0,01 for FK506, CyA, methylprednisolone and

rapamycin respectively) (figure 3, c).

An overview of all (adjusted) p values is given in table 1 (supplementary section).

Discussion

Humoral immune reactions can impair the long-term prognosis of solid organ

transplantation. Allo-antibodies and auto-antibodies to organ-specific self-antigens



13

might damage organ function because of their role in chronic graft rejection.

Furthermore, the development of de novo autoimmune diseases and allergic disorders

puts a burden on solid organ transplant patients. CNI are the immunosuppressive

drugs of choice after solid organ transplantation but their direct effect on the humoral

immune response is not well known. Hence, we examined the in vitro effect of

immunosuppression on differentiation and class-switching of total and naive B cells

and the effect of these immunosuppressive drugs on Tfh cell differentiation from

naive CD4+ T cells. We found that CNI do exert an effect on humoral immunity

directly by suppressing naive B cells and not only indirectly via their effect on T cell

help.

Tfh cells exert an important role in autoimmune disease. Increased frequencies of Tfh

cells have been associated with autoimmune thyroid disease, systemic lupus

erythematosus and systemic sclerosis 14-16

. CNI, rapamycin and methylprednisolone

inhibited Tfh cell differentiation from naive CD4+ T cells.

Hence, the development of auto-antibodies in solid organ transplant patients does not

seem a consequence of a direct effect of the immunosuppressive drugs on Tfh cells.

Our experiments were carried out in vitro on monocultures of either B or T cells. The

in vivo findings in solid organ transplant patients, however, can result from an overall

net effect of CNI on the interplay of B cells and different T cell subsets.

In total B cells, CNI did not affect B cell proliferation, plasmablast differentiation or

surface immunoglobulin expression. In naive B cells, however, CNI did inhibit B cell

proliferation and plasmablast differentiation. IgA was suppressed by both CNI

whereas IgE class-switching was suppressed by CyA only. These results demonstrate

a direct inhibitory effect of CNI on humoral immunity alongside of their influence on

T cell help, since in all conditions T cell help was provided.



14

CyA suppressed both IgE surface expression and secretion in supernatant of naive B

cells. With FK506, IgE surface expression even tended to rise, leaving IgE secretion

unaffected. This might explain the reported disappearance of clinical and biological

symptoms of newly acquired allergy post transplant after a switch from a FK506 to a

CyA based immunosuppressive regimen 30

. The suppression of IgE class-switching by

CyA, but not by FK506, might be responsible for this observation.

Heidt et al demonstrated that FK506, CyA and rapamycin attenuate cytokine

production (including IFN-γ) of activated CD4+

T cells on mRNA level 10

. In analogy

with our findings, Abadja et al described that FK506 caused a dramatic decrease of

IL-21 expression, as well as a less pronounced reduction of IFN-γ expression by

CD4+

T cells 12

. However no data are available on the effect of CNI on Tfh cell

differentiation, which might be of relevance in view of the increased auto-immunity

observed in solid organ transplant patients. We demonstrated that CNI did not have a

direct stimulating effect on Tfh cell differentiation from naive CD4+ T cells.

Furthermore, the number of IL-21+ Tfh cells decreased with increasing doses of

immunosuppression, which was most pronounced with CyA. An alternative that we

did not study here, is that CNI might affect the balance between Tfh and other T

helper cells 31,32

, which hence could influence humoral immunity.

Our in vitro experiments were carried out on PBMC from adult donors. The immune

system of neonates and young children is thought to be immature and more

tolerogenic compared to that of adults. Naive B and T cells better reflect this

immature immune system. The differential effect of CNI on naive versus total B cells

should therefore be taken in consideration when treating young patients, given the

possibility that immunosuppressive drugs such as FK506 could exhibit a specific

effect on the immature humoral immune system. This specific effect could play a role



15

in the observation that young children are more prone to develop allergic disorders

after transplantation. De novo acquired food allergy indeed is very common in

children after liver transplantation and rarely encountered in the adult transplant

population.

We showed that rapamycin had a strong dose-dependent inhibitory effect on B cell

number and proliferation in both total and naive B cells. This effect can be explained

by an inhibition of B cell cycle progression 33,34

but also by induction of B cell

apoptosis 35

. The potent inhibition of rapamycin on plasmablast differentiation and Ig

class-switching and Ig secretion we observed, is a logical downstream effect hereof.

Despite the long term use of glucocorticoids in B cell dependent diseases, their effects

on B cells have not been intensily investigated. B cells are still considered to be

relatively ‘resistant’ to glucocorticoids although glucocorticoid receptors have been

found on the B cell membrane of patients affected with rheumatoid arthritis 36

. It has

been shown that treatment with high doses of dexamethasone can cause a significant

reduction of circulating B cell activating factor (BAFF) and its mRNA 37

. BAFF

which is a member of the tumor necrosis factor (TNF) family, is capable of regulating

B lymphocyte survival and maturation, antibody production and immunoglobulin

switching 38,39

. Remarkably, we observed that especially medium concentrations of

methylprednisolone caused a significant increase in CD38+ plasmablast

differentiation from naive B cells in vitro. This finding is new and the mechanism

unclear. Methylprednisolone might have a stimulating effect on B cell activation

which only becomes apparent in naive B cells, by lowering their threshold for

activation. However, immunoglobulin secretion, did not follow the rise in

plasmablasts, so perhaps the development of these methylprednisolone-induced

plasmablasts into functional plasma cells was impaired.



16

Furthermore, methylprednisolone induced IgE class-switching in low and medium

concentrations in naive B cells. In analogy, Jabara et al also showed an induction of in

vitro IgE class-switching in human purified B cells by hydrocortisone 40

. A polyclonal

rise in serum IgE has also been seen in vivo in patients treated with prednison. These

findings, which seem to be in contrast with the clinically observed anti-allergic effects

of steroid therapy, in fact support a broader anti-inflammatory and

immunomodulatory effect by suppressing cytokine production and inflammatory cell

infiltration 41-43

.

Conclusion

B cell proliferation, plasmablast differentiation and surface immunoglobulin

expression are not directly affected by CNI in total B cells, whilst in naive B cells

CNI inhibit these processes. This differential effect might need to be taken into

consideration when treating young patients. Furthermore, CNI, as well as rapamycin

and methylprednisolone, inhibit in vitro differentiation of Tfh from naive CD4+ T

cells. In view of its potent suppressive effect on B cell division, plasmablast

differentiation, Ig secretion and Tfh cell differentiation, rapamycin might be an

interesting candidate for the prevention and/or treatment of B cell mediated

complications after solid organ transplantation.

Acknowledgements

We thank Prof Dr Karim Vermaelen for use of the laboratory infrastructure and Kim

Deswarte for his assistance with cell sorting.

Funding



17

P Gevaert, M Dullaers, R De Bruyne received a grant as respectively senior clinical

investigator, postdoctoral fellow and clinical Ph.D. fellow by the Flemish Scientific

Research Board (FWO Vlaanderen). The research was supported by a personal grant

of FWO to M Dullaers (FWO14/KAN/019).

Disclosure

All authors declare that they have no relevant conflicts of interest.

References

1. Sarma NJ, Tiriveedhi V, Angaswamy N, Mohanakumar T. Role of antibodies

to self-antigens in chronic allograft rejection: potential mechanism and

therapeutic implications. Hum Immunol. 2012;73(12):1275–1281.

doi:10.1016/j.humimm.2012.06.014.

2. Gupta P, Hart J, Millis JM, Cronin D, Brady L. De novo hepatitis with

autoimmune antibodies and atypical histology: a rare cause of late graft

dysfunction after pediatric liver transplantation. Transplantation.

2001;71(5):664–668.

3. Asante-Korang A, Boyle GJ, Webber SA, Miller SA, Fricker FJ. Experience of

FK506 immune suppression in pediatric heart transplantation: a study of long-

term adverse effects. J Heart Lung Transplant. 1996;15(4):415–422.

4. Gruber S, Tiringer K, Dehlink E, et al. Allergic sensitization in kidney-

transplanted patients prevails under tacrolimus treatment. Clinical &

Experimental Allergy. 2011;41(8):1125–1132. doi:10.1111/j.1365-

2222.2011.03761.x.

5. Kawamura N, Furuta H, Tame A, et al. Extremely high serum level of IgE

during immunosuppressive therapy: paradoxical effect of cyclosporine A and

tacrolimus. Int Arch Allergy Immunol. 1997;112(4):422–424.

6. Coelho T, Tredger M, Dhawan A. Current status of immunosuppressive agents

for solid organ transplantation in children. Pediatr Transplant.

2012;16(2):106–122. doi:10.1111/j.1399-3046.2012.01644.x.

7. Spada M, Riva S, Maggiore G, Cintorino D, Gridelli B. Pediatric liver

transplantation. World J Gastroenterol. 2009;15(6):648–674.



18

8. Saeed SA, Integlia MJ, Pleskow RG, et al. Tacrolimus-associated eosinophilic

gastroenterocolitis in pediatric liver transplant recipients: role of potential food

allergies in pathogenesis. Pediatr Transplant. 2006;10(6):730–735.

doi:10.1111/j.1399-3046.2006.00538.x.

9. Wiederrecht G, Lam E, Hung S, Martin M, Sigal N. The mechanism of action

of FK-506 and cyclosporin A. Ann N Y Acad Sci. 1993;696:9–19.

10. Heidt S, Roelen DL, Eijsink C, et al. Calcineurin inhibitors affect B cell

antibody responses indirectly by interfering with T cell help. Clin Exp

Immunol. 2010;159(2):199–207. doi:10.1111/j.1365-2249.2009.04051.x.

11. Kim HY, Cho M-L, Jhun JY, et al. The imbalance of T helper 17/regulatory T

cells and memory B cells during the early post-transplantation period in

peripheral blood of living donor liver transplantation recipients under

calcineurin inhibitor-based immunosuppression. Immunology.

2013;138(2):124–133. doi:10.1111/imm.12021.

12. Abadja F, Atemkeng S, Alamartine E, Berthoux F, Mariat C. Impact of

mycophenolic acid and tacrolimus on Th17-related immune response.

Transplantation. 2011;92(4):396–403. doi:10.1097/TP.0b013e3182247b5f.

13. Fazilleau N, Mark L, McHeyzer-Williams LJ, McHeyzer-Williams MG.

Follicular helper T cells: lineage and location. Immunity. 2009;30(3):324–335.

doi:10.1016/j.immuni.2009.03.003.

14. Zhu C, Ma J, Liu Y, et al. Increased frequency of follicular helper T cells in

patients with autoimmune thyroid disease. J Clin Endocrinol Metab.

2012;97(3):943–950. doi:10.1210/jc.2011-2003.

15. Simpson N, Gatenby PA, Wilson A, et al. Expansion of circulating T cells

resembling follicular helper T cells is a fixed phenotype that identifies a subset

of severe systemic lupus erythematosus. Arthritis Rheum. 2010;62(1):234–244.

doi:10.1002/art.25032.

16. Morita R, Schmitt N, Bentebibel S-E, et al. Human blood CXCR5(+)CD4(+) T

cells are counterparts of T follicular cells and contain specific subsets that

differentially support antibody secretion. Immunity. 2011;34(1):108–121.

doi:10.1016/j.immuni.2010.12.012.

17. Crotty S. Follicular helper CD4 T cells (TFH). Annu Rev Immunol.

2011;29:621–663. doi:10.1146/annurev-immunol-031210-101400.

18. Kato LM, Kawamoto S, Maruya M, Fagarasan S. Gut TFH and IgA: key

players for regulation of bacterial communities and immune homeostasis.

Immunology and Cell Biology. 2014;92(1):49–56. doi:10.1038/icb.2013.54.

19. Breitfeld D, Ohl L, Kremmer E, et al. Follicular B helper T cells express CXC

chemokine receptor 5, localize to B cell follicles, and support immunoglobulin

production. J Exp Med. 2000;192(11):1545–1552.

20. King C, Tangye SG, Mackay CR. T follicular helper (TFH) cells in normal and



19

dysregulated immune responses. Annu Rev Immunol. 2008;26:741–766.

doi:10.1146/annurev.immunol.26.021607.090344.

21. Moser B, Schaerli P, Loetscher P. CXCR5(+) T cells: follicular homing takes

center stage in T-helper-cell responses. Trends Immunol. 2002;23(5):250–254.

22. Vogelzang A, McGuire HM, Yu D, Sprent J, Mackay CR, King C. A

fundamental role for interleukin-21 in the generation of T follicular helper

cells. Immunity. 2008;29(1):127–137. doi:10.1016/j.immuni.2008.06.001.

23. Liu YJ, Arpin C. Germinal center development. Immunol Rev. 1997;156:111–

126.

24. MacLennan IC. Germinal centers. Annu Rev Immunol. 1994;12(1):117–139.

doi:10.1146/annurev.iy.12.040194.001001.

25. Dullaers M, Li D, Xue Y, et al. A T cell-dependent mechanism for the

induction of human mucosal homing immunoglobulin A-secreting

plasmablasts. Immunity. 2009;30(1):120–129.

doi:10.1016/j.immuni.2008.11.008.

26. Schaerli P, Willimann K, Lang AB, Lipp M, Loetscher P, Moser B. CXC

chemokine receptor 5 expression defines follicular homing T cells with B cell

helper function. J Exp Med. 2000;192(11):1553–1562.

27. Coffman RL, Lebman DA, Shrader B. Transforming growth factor beta

specifically enhances IgA production by lipopolysaccharide-stimulated murine

B lymphocytes. J Exp Med. 1989;170(3):1039–1044.

28. Lebman DA, Coffman RL. Interleukin 4 causes isotype switching to IgE in T

cell-stimulated clonal B cell cultures. J Exp Med. 1988;168(3):853–862.

29. Schmitt N, Morita R, Bourdery L, et al. Human dendritic cells induce the

differentiation of interleukin-21-producing T follicular helper-like cells through

interleukin-12. Immunity. 2009;31(1):158–169.

doi:10.1016/j.immuni.2009.04.016.

30. Maarof G, Krzysiek R, Décline J-L, Cohen J, Habes D, Jacquemin E.

Management of post-liver transplant-associated IgE-mediated food allergy in

children. J Allergy Clin Immunol. 2011;127(5):1296–1298.

doi:10.1016/j.jaci.2010.12.1094.

31. Kim H-P, Korn LL, Gamero AM, Leonard WJ. Calcium-dependent activation

of interleukin-21 gene expression in T cells. J Biol Chem.

2005;280(26):25291–25297. doi:10.1074/jbc.M501459200.

32. Nakayamada S, Takahashi H, Kanno Y, O'Shea JJ. Helper T cell diversity and

plasticity. Curr Opin Immunol. 2012;24(3):297–302.

doi:10.1016/j.coi.2012.01.014.

33. Aagaard-Tillery KM, Jelinek DF. Inhibition of human B lymphocyte cell cycle

progression and differentiation by rapamycin. Cell Immunol. 1994;156(2):493–



20

507. doi:10.1006/cimm.1994.1193.

34. Zhang S, Readinger JA, DuBois W, et al. Constitutive reductions in mTOR

alter cell size, immune cell development, and antibody production. Blood.

2011;117(4):1228–1238. doi:10.1182/blood-2010-05-287821.

35. Heidt S, Roelen DL, Eijsink C, van Kooten C, Claas FHJ, Mulder A. Effects of

immunosuppressive drugs on purified human B cells: evidence supporting the

use of MMF and rapamycin. Transplantation. 2008;86(9):1292–1300.

doi:10.1097/TP.0b013e3181874a36.

36. Bartholome B, Spies CM, Gaber T, et al. Membrane glucocorticoid receptors

(mGCR) are expressed in normal human peripheral blood mononuclear cells

and up-regulated after in vitro stimulation and in patients with rheumatoid

arthritis. FASEB J. 2004;18(1):70–80. doi:10.1096/fj.03-0328com.

37. Huard B, Schneider P, Mauri D, Tschopp J, French LE. T cell costimulation by

the TNF ligand BAFF. The Journal of Immunology. 2001;167(11):6225–6231.

38. Youinou P, Pers J-O. The late news on baff in autoimmune diseases.

Autoimmun Rev. 2010;9(12):804–806. doi:10.1016/j.autrev.2010.06.011.

39. Zen M, Canova M, Campana C, et al. The kaleidoscope of glucorticoid effects

on immune system. Autoimmun Rev. 2011;10(6):305–310.

doi:10.1016/j.autrev.2010.11.009.

40. Jabara HH, Ahern DJ, Vercelli D, Geha RS. Hydrocortisone and IL-4 induce

IgE isotype switching in human B cells. J Immunol. 1991;147(5):1557–1560.

41. Zieg G, Lack G, Harbeck RJ, Gelfand EW, Leung DY. In vivo effects of

glucocorticoids on IgE production. J Allergy Clin Immunol. 1994;94(2 Pt

1):222–230.

42. Settipane GA, Pudupakkam RK, McGowan JH. Corticosteroid effect on

immunoglobulins. J Allergy Clin Immunol. 1978;62(3):162–166.

43. Posey WC, Nelson HS, Branch B, Pearlman DS. The effects of acute

corticosteroid therapy for asthma on serum immunoglobulin levels. J Allergy

Clin Immunol. 1978;62(6):340–348.



21

Figure legends

Figure 1

Influence of immunosuppression on B cell division and differentiation in vitro.

Total and naive B cells were cultured for 7 days with or without immunosuppressive

drugs (IS) in IgG- / IgE- (IL-4) and IgA- (TGF-β) promoting conditions. Proliferation

and CD38+ plasmablast differentiation were analyzed using flow cytometry. Mean ±

SD of 5 different healthy donors without or with titrated doses of IS are plotted for

CD19+ alive B cell count (a, b), percentage of divided cells (CFSE) (c, d) and

percentage of CD38+ plasmablasts (e, f). Per donor, the measured values were divided

by the value of the condition without IS (‘none’), to compensate for inter-donor

variability. Rapa, rapamycin; MethylPred, methylprednisolone; FK506, tacrolimus;

CyA, cyclosporine A.

Figure 2

Influence of immunosuppression on immunoglobulin class-switching and

secretion in vitro.

Total and naive B cells were cultured for 7 days with or without immunosuppressive

drugs (IS) in IgG- / IgE- (IL-4) and IgA- (TGF-β, CpG) promoting conditions.

Surface Ig expression were analyzed by flow cytometry. Mean ± SD of 5 different

healthy donors without or with titrated doses of IS are plotted for IgE and IgA surface

staining (a, b). Secreted IgE, IgA and IgG were measured by ELISA in supernatant

and the mean ± SD of 5 different healthy donors was plotted (c, d). Per donor, the

measured values were divided by the value of the condition without IS (‘none’), to



22

compensate for interdonor variability. Rapa, rapamycin; MethylPred,

methylprednisolone; FK506, tacrolimus; CyA, cyclosporine A.

Figure 3

Influence of immunosuppression on T follicular helper (Tfh) cell differentiation

from naive CD4+ T cells in vitro.

Naive CD4+ T cells were stimulated with anti-CD3, anti-CD28 and IL-12 for 48 hours

to induce Tfh cell differentiation with or without immunosuppressive drugs (IS).

Interferon-γ (INF-γ) and IL-21 were analyzed by intracellular cytokine staining (a)

and ELISA on the culture supernatant (c). Mean ± SD of 6 different healthy donors

are plotted. T cell division was monitored using Cell trace Violet (CTV) dilution (b).

Absolute values were per donor normalized to the condition without IS (‘none’), to

compensate for interdonor variability. Rapa, rapamycin; MethylPred,

methylprednisolone; FK506, tacrolimus; CyA, cyclosporine A.

Supplementary figure 1

Isotype skewing by different cytokines in human naive B cells.

Naive B cells were cultured for 7 days in the presence of anti-CD40 antibody and IL-

2 alone or IL-2+IL-21 or IL-2+IL-21+TGF-β1 or IL-2+IL-21+IL-4 as indicated.

Secreted IgA, IgE and IgG were measured by ELISA in supernatants and the mean ±

SD of 9 different healthy donors was plotted. Raw data are shown.



b

Naive B cells Total B cells IL-4 TGF-β IL-4 TGF-β

d

f

None Low Medium High0

1

2

3

4

5

Concentration IS

CD19+ alive B cells

None Low Medium High0.0

0.5

1.0

1.5

2.0

Concentration IS

divided cells


0.5

1.0

1.5

2.0

Concentration IS

CD38+ plasmablasts


0.5

1.0

1.5

2.0

Concentration IS

Cel

l num

ber /

no

IS

CD19+ alive B cells


0.5

1.0

1.5

2.0

Concentration IS

% d

ivid

ed c

ells

/ no

IS

divided cells


1

2

3

Concentration IS

% C

D38

+ / n

o IS

CD38+ plasmablastsh


1

2

3

4

5

Concentration ISC

ell n

umbe

r / n

o IS

CD19+ alive B cells


0.5

1.0

1.5

2.0

Concentration IS

% d

ivid

ed c

ells

/ no

ISdivided cells


0.5

1.0

1.5

2.0

Concentration IS

% C

D38

+ / n

o IS

CD38+ plasmablasts

d

f


0.5

1.0

1.5

2.0

Concentration IS

Cel

l num

ber /

no

IS

CD19+ alive B cells


0.5

1.0

1.5

2.0

Concentration IS

% d

ivid

ed c

ells

/ no

IS

divided cells


1

2

3

Concentration IS

% C

D38

+ / n

o IS

CD38+ plasmablasts

CyAFKMPrapa


4445464748495051525354555657585960



1

2

3

Concentration IS

Secr

eted

IgE

/ no

IS

IgE

Naive B cells Total B cells


1

2

3

Concentration IS

% o

f aliv

e ce

lls /

no IS

IgE+ cells

IL-4 IL-4 TGF-β TGF-β

IgG


1

2

3

Concentration IS

Secr

eted

IgG

/ no

IS

IgE


1

2

3

Concentration IS

Secr

eted

IgE

/ no

IS

IgG


1

2

3

Concentration IS

Secr

eted

IgG

/ no

IS


1

2

3

Concentration IS

% o

f aliv

e ce

lls /

no IS

IgE+ cells


1

2

3

Concentration IS

% o

f aliv

e ce

lls /

no IS

IgA+ cells

b


1

2

3

Concentration IS

Secr

eted

IgA

/ no

IS

IgA IgA


1

2

3

Concentration IS

Secr

eted

IgA

/ no

IS

CyAFKMPrapa


1

2

3

Concentration IS

% o

f aliv

e ce

lls /

no IS

IgA+ cells

d


4445464748495051525354555657585960


CyAFKMPrapa


0.5

1.0

1.5

2.0

Concentration IS

% o

f CD

4+ T

cells

/ no

IS INFγ+


0.5

1.0

1.5

2.0

Concentration IS%

of C

D4+

T ce

lls/ n

o IS

IL-21+


0.5

1.0

1.5

2.0

Concentration IS

% o

f CD

4+ T

cells

/ no

IS IL-21+ INFγ+


0.5

1.0

1.5

2.0

Concentration IS

% o

f aliv

e ce

lls /

no IS

CD4+ T cell division


0.5

1.0

1.5

2.0

Concentration IS

Secr

eted

IFN

-γ /

no IS

Secreted INF-γ

c


0.5

1.0

1.5

2.0

Concentration IS

Secr

eted

IL-2

1 / n

o IS

Secreted IL-21


4445464748495051525354555657585960


IgA

IL-2

IL-2+IL-21

IL-2+IL-21+IL-4

IL-2+IL-21+TGF-β1

0

100

200

300

ng/mL

IgE

IL-2

IL-2+IL-21

IL-2+IL-21+IL-4

IL-2+IL-21+TGF-β1

0

5

10

15

20

ng/mL

IgG

IL-2

IL-2+IL-21

IL-2+IL-21+IL-4

IL-2+IL-21+TGF-β1

0

100

200

300

400

500

ng/mL


4445464748495051525354555657585960


Supplementary table 1

Overview of (adjusted) p values for all examined conditions.

Friedman

test

Multiple comparisons

none

versus

low

none

versus

medium

none

versus

high

B cell number

Total B cells

+ IL-4 FK 0,06 ns ns ns

CyA 0,04 ns ns ns

MP < 0,001 ns 0,04 0,002

RAPA < 0,001 ns ns < 0,001

Total B cells

+ TGF-β

FK 0,002 ns ns < 0,001

CyA < 0,001 ns 0,02 < 0,001

MP 0,3 ns ns ns

RAPA < 0,001 ns 0,01 < 0,001

Naive B cells

+ IL-4

FK 0,7 ns ns ns

CyA 0,05 ns ns ns

MP 0,05 ns ns ns

RAPA 0,4 ns ns ns

Naive B cells

+ TGF-β

FK 0,02 ns ns 0,01

CyA 0,002 ns ns 0,01

MP 0,9 ns ns ns

RAPA 0,002 ns ns ns

B cell division

Total B cells


CyA 0,4 ns ns ns

MP 0,03 ns ns ns

RAPA < 0,001 ns 0,02 < 0,001

Total B cells

+ TGF-β

FK 0,2 ns ns ns

CyA 0,05 ns ns 0,04

MP 0,3 ns ns ns

RAPA < 0,001 ns 0,002 < 0,001

Naive B cells

+ IL-4

FK 0,7 ns ns ns

CyA 0,02 ns ns ns

MP 0,07 ns ns ns



RAPA < 0,001 ns 0,02 0,009

Naive B cells

+ TGF-β

FK < 0,001 ns ns 0,002

CyA 0,02 ns ns ns

MP 0,3 ns ns ns

RAPA < 0,001 ns ns 0,04

CD38+ B cells

Total B cells


CyA 0,3 ns ns ns

MP 0,05 ns ns ns

RAPA < 0,001 ns ns < 0,001

Total B cells

+ TGF-β

FK 0,1 ns ns ns

CyA 0,05 ns ns ns

MP 0,2 ns ns ns

RAPA < 0,001 ns 0,02 < 0,001

Naive B cells

+ IL-4

FK 0,02 ns ns 0,02

CyA 0,02 ns 0,004 ns

MP 0,007 ns 0,009 ns

RAPA < 0,001 ns 0,04 < 0,001

Naive B cells

+ TGF-β

FK < 0,001 ns 0,04 0,004

CyA 0,02 ns ns 0,03

MP 0,001 ns 0,004 ns

RAPA < 0,001 ns ns 0,002

IgE+ B cells

Total B cells FK 0,2 ns ns ns

CyA 0,2 ns ns ns

MP 0,02 ns ns 0,005

RAPA < 0,001 ns 0,001 < 0,001

Naive B cells

FK 0,9 ns ns ns

CyA 0,02 ns ns ns

MP 0,003 0,03 0,02 ns

RAPA < 0,001 ns ns 0,02

IgA+ B cells


CyA 0,6 ns ns ns

MP 0,002 ns ns < 0,001

RAPA < 0,001 ns ns 0,02

Naive B cells

FK < 0,001 ns ns 0,002

CyA 0,002 ns ns 0,01

MP 0,002 ns ns 0,04

RAPA < 0,001 ns 0,04 < 0,001



IgE in SN


CyA 0,08 ns ns ns

MP 0,08 ns ns ns

RAPA 0,07 ns ns ns

Naive B cells

FK 0,5 ns ns ns

CyA 0,04 ns ns ns

MP 0,02 ns ns ns

RAPA 0,001 ns ns 0,01

IgA in SN

Total B cells FK 0,002 ns ns 0,003

CyA 0,007 ns ns 0,01

MP 0,5 ns ns ns

RAPA < 0,001 ns < 0,001 < 0,001

Naive B cells

FK 0,02 ns ns 0,01

CyA 0,04 ns ns ns

MP 0,07 ns ns ns

RAPA 0,02 ns 0,02 0,03

IgG in SN

(IL-4)


CyA 0,42 ns ns ns

MP 0,8 ns ns ns

RAPA < 0,001 ns ns < 0,001

Naive B cells

FK 0,34 ns ns ns

CyA 0,05 ns ns ns

MP 0,61 ns ns ns

RAPA 0,01 ns 0,06 ns

CD4+ cells

INF-γ+

FK < 0,001 ns 0,04 < 0,001

CyA < 0,001 ns ns 0,002

MP < 0,001 ns ns 0,04

RAPA < 0,001 ns 0,01 0,001

IL-21+

FK < 0,001 ns 0,01 < 0,001

CyA < 0,001 ns 0,002 0,002

MP 0,003 ns ns 0,01

RAPA 0,003 ns ns 0,005

INF-γ+ IL-21

+

FK < 0,001 ns 0,005 0,001

CyA < 0,001 ns 0,005 0,001

MP < 0,001 ns ns 0,005

RAPA < 0,001 ns 0,02 0,001

Secreted INF-γ

FK < 0,001 ns ns < 0,001

CyA < 0,001 ns 0,02 < 0,001

MP < 0,001 ns 0,04 < 0,001

RAPA < 0,001 ns 0,02 < 0,001



Secreted IL-21

FK < 0,001 0,01 ns 0,002

CyA 0,002 0,007 ns 0,01

MP 0,03 ns 0,01 ns

RAPA 0,01 ns 0,01 ns

T cell division

FK < 0,001 ns 0,04 < 0,001

CyA < 0,001 ns 0,003 0,01

MP < 0,001 ns ns 0,02

RAPA < 0,001 ns 0,01 < 0,001



Supplementary table 1:

(Adjusted) p values after Friedman test and Dunn’s multiple comparisons test for all

examined conditions (each compared to the same condition without immunosuppression).

A(n) (adjusted) p value of ≤ 0,05 was considered statistically significant. CyA, cyclosporine

A; FK, tacrolimus; MP, methylprednisolone; ns, non significant; RAPA, rapamycin; SN,

supernatant.



Calcineurin inhibitors dampen humoral immunity by acting directly on naive B cells

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