bongha shin transplantation paper

Regulation of Anti-HLA Antibody-Dependent NaturalKiller Cell Activation by Immunosuppressive Agents

Bong-Ha Shin,1,4 Shili Ge,1 James Mirocha,3 Artur Karasyov,1 Ashley Vo,2 Stanley C. Jordan,2

and Mieko Toyoda1

Background. It was demonstrated that human natural killer (NK) cells, via antibody-dependent cellular cytotoxicity(ADCC)-like mechanism, increase IFNF production after exposure to alloantigens. This finding was associated withan increased risk for antibody-mediated rejection (ABMR). Although the effects of various immunosuppressive drugson T cells and B cells have been extensively studied, their effects on NK cells are less clear. This study reports the effectof immunosuppressive agents on antibody-mediated NK cell activation in vitro.Methods. Whole blood from normal individuals was incubated with irradiated peripheral blood mononuclear cells(PBMCs) pretreated with anti-HLA antibody+ sera (in vitro ADCC), with or without immunosuppressive agents.The %IFNF+ and CD107a+ (degranulation marker) in CD56+ NK cells were enumerated by flow cytometry.Results. Cyclosporine A and tacrolimus significantly reduced IFNF production in a dose-dependent manner(53%Y83%), but showed minimal effect on degranulation (20%). Prednisone significantly reduced both IFNF pro-duction and degranulation (50%Y66% reduction at maximum therapeutic levels). Calcineurin inhibitors (CNIs) incombination with prednisone additively suppressed IFNF production and degranulation. The effect of sirolimus ormycophenolate mofetil on NK cells was minimal.Conclusions. These results suggest that potent suppressive effects of CNIs and prednisone on antibody-mediated NKcell activation may contribute to the reduction of ADCC in sensitized patients and possibly reduce the risk for ADCC-mediated ABMR. These further underscore the importance of medication compliance in prevention of ABMR andpossibly chronic rejection, and suggest that ADCC-mediated injury may increase in strategies aimed at CNI or steroidminimization or avoidance.

Keywords: NK cells, IFNF, CD107a, Immunosuppressive drugs, Antibody-dependent cellular cytotoxicity.

(Transplantation 2014;97: 00Y00)

Antibody-mediated rejection (ABMR) is a major ob-stacle to successful transplantation in HLA-sensitized

(HS) patients. The traditional view of ABMR is that ofcomplement-dependent cytotoxicity (CDC)-mediated injurywith characteristic C4d deposition (1, 2). However, cellular

effector pathways including antibody-dependent cellular cyto-toxicity (ADCC) may also play an important role in the path-ogenesis of ABMR (3Y6). The authors previously reported onthe development of the allo-CFC assay to measure CD3j(non-T) cell response to alloantigens expressed on peripheralblood mononuclear cells (PBMCs) by detecting intracellu-lar IFNF production. Using this assay, it was found thatalloantigen-specific CD3j cells were elevated in most HS pa-tients, but not in normal individuals without history of allo-antigen exposure. HS patients who were allo-CFC high (+)pretransplant were at high risk for ABMR after transplant(7, 8). In a subsequent study, it was found that IFNF+/CD3jcells in response to allo-PBMC detected in the allo-CFC assaywere natural killer (NK) cells and this NK cell activation wasantibody-mediated via FcFRIIIa (CD16) on NK cells, which isan ADCC-like mechanism (9). These results suggest that NKcell activation via ADCC is likely important in mediatingABMR and may represent a newly recognized opportunity formodification of antibody-mediated allograft injury.

Among various immunosuppressive drugs currentlyused in transplant recipients, calcineurin inhibitors (CNIsVcyclosporine A [CSA], tacrolimus [TAC]), mycophenolatemofetil (MMF), sirolimus (SRL) and corticosteroid, and thecombinations of these drugs are common. The effects of these

BASIC AND EXPERIMENTAL RESEARCH

Transplantation & Volume 97, Number 3, February, 2014 www.transplantjournal.com 1

The authors declare no funding or conflicts of interest.1Transplant Immunology Laboratory, Comprehensive Transplant Center,

Cedars-Sinai Medical Center/UCLA School of Medicine, Los Angeles, CA.2Comprehensive Transplant Center, Cedars-Sinai Medical Center/UCLA

School of Medicine, Los Angeles, CA.3Biostatistics Core, Research Institute, General Clinical Research Center,

Cardiothoracic Surgery, Cedars-Sinai Medical Center, Los Angeles, CA.4Address correspondence to: Bong-Ha Shin, Ph.D., Transplant Immunology

Laboratory, SSB111, Comprehensive Transplant Center, Cedars-SinaiMedical Center, 8700 Beverly Blvd., Los Angeles, CA 90048.

E-mail: [email protected]. participated in designing and performing the research, analyzing the

data, and writing the article. S.G. participated in performing the research.J.M. participated in analyzing the data. A.K. participated in performingthe research. A.V. participated in performing the research. S.C.J. par-ticipated in writing the article. M.T. participated in designing the re-search, analyzing the data, and writing the article.

Received 01 August 2013. Revision requested 16 October 2013.Accepted 22 October 2013.Copyright * 2013 by Lippincott Williams & WilkinsISSN: 0041-1337/14/9703-00DOI: 10.1097/01.TP.0000438636.52085.50

Copyright © 2013 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

mailto:[email protected]

drugs on T cells and B cells have been extensively studied (10).However, studies investigating regulatory effects on NK cells,especially antibody-mediated NK cell activation, are limited.Upon activation of NK cells via ADCC, NK cells release a pro-inflammatory cytokine, IFNF, as well as perforin and granzymeB through degranulation, resulting in killing of target cells.Degranulation results in appearance of inner lysosomal mem-brane protein, CD107a, on the surface of NK cells, which isused for assessment of NK cell degranulation (11). This studyestablished an in vitro alloantigen-dependent ADCC systemand examined the effect of various immunosuppressive drugson antibody-mediated NK cell activation by assessing IFNF

production and degranulation in NK cells.

RESULTS

The Effect of CSA, TAC, Prednisone, SRL, andMMF on IFNF Production and Degranulation inNK Cells Activated Via In Vitro ADCC

A typical result in the in vitro ADCC is shown inFigure 1. Significant increase of IFNF production and de-granulation as assessed by surface CD107a expression onNK cells were observed in the in vitro ADCC compared tocontrols without alloantibody-coated PBMCx stimulation

(Figs. 1A, B). The average IFNF+% and CD107a+% inCD56+ NK cells in the ADCC condition included in thisstudy, 22.9 and 20.8, were significantly higher than 0.8 and1.6 in control, respectively. Using this in vitro ADCC, theeffect of immunosuppressive drugs on alloantibody-mediatedNK cell activation was assessed.

CSA and TAC significantly suppressed IFNF produc-tion in a dose dependent manner, while they showed a mildsuppressive effect on degranulation (Figs. 2A, B). The re-duction rate of IFNF production was 30% by CSA and 25%by TAC at the trough levels (300 ng/mL and 15 ng/mL, re-spectively), and reached at 90% and 35% at the highesttherapeutic levels (1,000 ng/mL and 70 ng/mL, respectively).In contrast, the reduction of degranulation by CSA and TACwas only 20% and 15%, respectively, at the highest thera-peutic levels, and was not statistically significant. Prednisonesignificantly suppressed both IFNF production and de-granulation in a dose-dependent manner (Fig. 2C). Thereduction rate was 66% and 50%, respectively, at the highesttherapeutic level (10,000 ng/mL). SRL and MMF showedminimal effect (15% reduction at most) on both IFNF

production and degranulation at the highest therapeutic levels(50 ng/mL and 10,000 ng/mL, respectively) (Figs. 2D, E).

FIGURE 1. IFNF production and degranulation in NK cells activated via in vitro ADCC. Typical negative (A) and positive(B) results in the in vitro ADCC using normal and HS patient sera, respectively, are shown. Lymphocytes first gated byforward/side scatter (a) were further plotted against CD3 and SSC (b). CD3j cells including NK cells were further plottedagainst CD56 and IFNF (c) or CD107a (d), and IFNF+ and CD107a+ cell% in CD56+ NK cells were calculated. Mean valuesof IFNF+ and CD107a+ cell% in CD56+ NK cells in the in vitro ADCC using HS sera without drug were 22.9 T 5.9 and 20.8 T6.4, respectively.

2 www.transplantjournal.com Transplantation & Volume 97, Number 3, February, 2014


The Effect of Combination of CNI and Prednisoneon IFNF Production and Degranulation in NKCells Activated Via In Vitro ADCC

Because the combination of CNI and corticosteroids iscommonly used in transplant recipients and both immu-nosuppressive drugs alone showed a suppressive effect onthe in vitro ADCC, the effect of combination of these drugswas next examined. CSA (Fig. 3) or TAC (Fig. 4) in com-bination with prednisone additively reduced IFNF produc-tion and degranulation in antibody-activated NK cells, butnot synergistically.

DISCUSSIONCurrent immunosuppressive protocols used for clini-

cal transplantation primarily target T cells (10). However,the effects of current maintenance immunosuppressiveagents on other immune cells such as NK cells are limited.NK cells have gained specific interest in organ transplanta-tion because of its ability to recognize MHC antigens, whichregulates NK cell activation and cytotoxicity (12). The NKcell capacity for immunological memory (13, 14) and itsprofound role in tolerance induction (15, 16) have recently

been suggested. The involvement of NK cells in chronic re-jection has been reported using microarray analysis of renalcortex tissue obtained from patients undergoing chronicrejection, most secondary to non-compliance (17, 18). Sev-eral other studies (19Y21), including the authors’ (7Y9), havealso suggested the involvement of NK cells in acute andchronic ABMR in HS transplant patients. Here, it wassuggested that NK cell activation via ADCC mechanism islikely important in mediating ABMR. Thus, it is of interestto investigate the effects of currently used immunosup-pressive agents for transplant recipients on ADCC-mediatedNK cell activation in vitro. This study first established an invitro antibody-mediated NK cell activation system (in vitroADCC) where NK cells from normal individuals were mixedwith anti-HLA antibody-coated allo-PBMCs, resulting inIFNF release and degranulation. Then, using this system, theeffect of various immunosuppressive agents on antibody-mediated NK cell activation was assessed. In this study,CNIs (CSA, TAC) and prednisone showed a dose-dependentinhibition of IFNF production in NK cells activated viain vitro ADCC. Prednisone also showed a dose-dependentinhibition of degranulation in activated NK cells, while the

FIGURE 2. The effect of CSA (A), TAC (B), prednisone (C), SRL (D), and MMF (E) on IFNF production and degranulation inNK cells activated via in vitro ADCC. The results are expressed as a percentage of IFNF+ (solid line) or CD107a+ (dottedline) cell% in CD56+ NK cells in the control condition without drug (100%). MeanTstandard deviation (SD) of five exper-iments is shown. Mean values of IFNF+ or CD107a+ cell% in CD56+ NK cells in the controls without additives were 21.0T4.8and 19.4T7.7, respectively. Shaded area represents a possible therapeutic drug concentration range. *PG0.0125 vs. controlwithout drug for prednisone and SRL, PG0.01 vs. control without drug for CSA, TAC, and MMF.

* 2013 Lippincott Williams & Wilkins Shin et al. 3


FIGURE 3. The effect of combination of CSA and prednisone on IFNF production (A) and degranulation (B) in NK cellsactivated via in vitro ADCC. The results are expressed as a percentage of IFNF+ cell% (A) and CD107a+ cell% (B) in CD56+NK cells in the control condition without drug (100%). MeanTstandard deviation (SD) of five experiments is shown. Meanvalues of IFNF+ cell% and CD107a+ cell% in CD56+ NK cells in controls without drug were 19.9T6.1 and 20.1T5.0, re-spectively. Shaded area represents a possible therapeutic prednisone concentration range. *PG0.0125 vs. without predni-sone at each CSA concentration, #PG0.0167 vs. without CSA at each prednisone concentration.

FIGURE 4. The effect of combination of TAC and prednisone on IFNF production (A) and degranulation (B) in NK cellsactivated via in vitro ADCC. The results are expressed as a percentage of IFNF+ cell% (A) and CD107a+ cell% (B) in CD56+NK cells in the control condition without drug (100%). MeanTstandard deviation (SD) of five experiments is shown. Meanvalues of IFNF+ cell% and CD107a+ cell% in CD56+ NK cells in controls without drug were 19.9T6.1 and 20.1T5.0, re-spectively. Shaded area represents a possible therapeutic prednisone concentration range. *PG0.0125 vs. without predni-sone at each TAC concentration, #PG0.0167 vs. without TAC at each prednisone concentration.



inhibition by CNIs was less than prednisone. When CNIs werecombined with prednisone, IFNF production and degranu-lation were suppressed additively, but not synergistically.

NK cellYmediated ADCC in humans is initiated by theengagement of low-affinity FCFRIIIA expressed on NK cellswith the Fc portion of antibodies bound to the target cellantigens (22Y24). FCFRIIIA is a multimeric receptor com-plex consisting of the ligand binding > subunit (CD16) as-sociated non-covalently with homodimers or heterodimersof X and F. Upon engagement, immunoreceptor tyrosine-based activation motifs in F subunit rapidly activates in-tracellular protein tyrosine kinases (PTKs) (25), which leadsto phosphorylation of phospholipase C, resulting in pro-duction of inositol triphosphate (IP3) and diacylglycerol(DAG). IP3 increases cytoplasmic calcium concentration,which induces calcineurin (CN)-dependent dephosphory-lation of nuclear factor of activated T cells (NFAT) (26),resulting in translocation of NFAT to the nucleus followedby binding to the promoter region of cytokine genes in-cluding INFF and transcribing those genes (27). On theother hand, DAG activates protein kinase C (PKC), whichinduces cytotoxic granule release in a calcium-dependentmanner (24). Another signal is transduced upon the en-gagement of FCFRIIIA with Fc portion of antibodies. Acti-vated intracellular PTKs phosphorylate phosphatidyl inositol3 kinase (PI3K) that then activates Rho-family GTPaseslinked to p21-activated kinase (PAK). Activated PAK thenphosphorylates MEK followed by ERK activation, whichresults in cytokine gene expression and cytotoxic granulerelease (28).

CNIs, CSA and TAC, are known to bind to cyclophilinand FKBP12, respectively, and inhibit CN-dependent de-phosphorylation of NFAT, resulting in inhibition of cyto-kine gene expression. As expected, both CSA and TACsignificantly inhibited IFNF production in NK cells stimu-lated via in vitro ADCC at possible therapeutic levels in adose-dependent manner. CSA especially showed 90% inhi-bition, suggesting that IFNF production induced via ADCCin NK cells is primarily through the CN-NFAT pathway andminimal through the PI3K pathway. As mentioned above,calcium-dependent PKC and ERK activations, but not CNpathway, are known to cause NK cell degranulation uponengagement of FCFRIIIA with Fc portion of antibodies.However, CNIs, especially CSA, showed approximately 20%reduction of degranulation at the highest possible thera-peutic level in this study. This suggests that CSA or CNIsmay have some effect on other signal transduction than theCN pathway. In fact, Morteau et al. (29) and Neudoerfl et al.(30) have recently reported that CNIs inhibited NK celldegranulation as well as IFNF production although theirexperimental systems were not ADCC setting.

The therapeutic and prophylactic use of prednisone iswidespread because of its powerful anti-inflammatory, anti-proliferative, and immunomodulatory activity. It has beenreported that prednisone inhibits transcriptional activityof pro-inflammatory cytokines including IFNF, adhesionmolecules, and enzymes such as Cox-2 and iNOS (31). Inthis study, prednisone showed a significant inhibitory effecton both IFNF production and degranulation at possibletherapeutic levels in a dose-dependent manner in NK cellsstimulated via in vitro ADCC. Although the mechanism

responsible for the inhibitory effect of prednisone onADCC-mediated NK cell activation is still not defined, it ispossible that prednisone inhibition is through glucocorti-coid receptors expressed on NK cells (32). In addition, otherreported studies are consistent with the authors’ own stud-ies. Nair et al (33) showed that prednisone significantlyinhibited NK cellYmediated ADCC in a dose-dependentmanner using peripheral blood NK cells reactive with ahuman B-cell leukemia cell line coated with rabbit anti-SBserum. They suggested that the inhibition by prednisone isa result of the reduction of the target-binding capacity ofNK cells by prednisone. Significant reduction of NK cellcytotoxicity by prednisone has also been shown in bonemarrow transplant patients who were treated with predni-sone for graft-versus-host disease (34), but the NK cyto-toxicity tested in their study was not via ADCC.

SRL, also known as rapamycin, binds to FKBP12 andthe SRL-FKBP12 complex in the cytoplasm of target cellsinhibits the kinase activity of mammalian target of ra-pamycin, which results in cell cycle arrest in the G1 to Stransition (35). However, the SRL-FKBP12 complex has noeffect on CN activity (36), which is consistent with the re-sults. In this study, SRL had no or minimum effect on IFNF

production or degranulation in NK cells in the in vitroADCC. It has been reported that SRL arrests cell cycle pro-gression in NK cells, but it does not seem to influence IFNF

production or cytotoxic activity using an in vitro system (37).Gourlay et al. (38) have shown that treatment of rats receiv-ing hamster skin grafts with SRL markedly diminishedthe production of anti-hamster cytotoxic antibodies, but itslightly enhanced NK cell function. Although the studies werenot performed in the ADCC setting, they suggested that theproliferation of NK cells might be regulated by SRL but cy-tokine production or cytotoxic activity of NK cells may not beinfluenced by SRL.

MMF also showed no or minimum effect on IFNF

production or degranulation in NK cells activated via the invitro ADCC. MMF is a pro-drug of mycophenolic acid, whichinhibits inosine monophosphate dehydrogenase (IMPDH),the rate-limiting enzyme of the de novo pathway of guaninesynthesis, which leads to reduction of intracellular guaninesynthesis, resulting in impairment of activated lymphocyteproliferation (39). A recent study has shown that IMPDHinhibited by MMF can be bypassed by activation of a purinesalvage pathway, which in turn leads to a continuous re-plenishment of intracellular GTP (40). In their follow-upstudy in heart transplant patients treated with MMF (41),they showed that the proportion of NK cells increased overtime while the number of total leukocytes remained con-stant. This suggested possible availability of the purine sal-vage pathway in NK cells because T cells and B cells lack thispathway. Vacher-Coponat et al have also shown that the NKcell number was preserved in 91 kidney-transplant patientstreated with MMF/FK506 and suggested possible preserva-tion of NK cell cytotoxicity in their patients (42). Thesestudies indirectly support our finding, but did not directlyaddress the inhibitory effect of MMF on ADCC in NK cells.

High rates of ABMR remain an obstacle to successfultransplant in HS patients even if they are desensitized.Although CDC-mediated graft injuries are traditionallythought to be the major cause of ABMR, NK cellYmediated



ADCC likely plays an important role in the pathogenesisof ABMR in both the acute and chronic state. In addi-tion, IFNF produced by activated NK cells induce MHCclass I and II expression on endothelial cells, rendering themmore susceptible to immune cell attack and priming (43). Inthis study, it was demonstrated that CNIs and prednisonesuppress IFNF production and degranulation in antibody-activated NK cells, suggesting a possible benefit of thesedrugs for prevention or modulation of the ADCC pathwayof injury in allografts. Recent data from a large cohort offailed kidney allografts demonstrated the primacy of donor-specific antibody (DSA)Yinduced injury to allografts thatultimately resulted in allograft loss (44, 45). The dogma formany years was that CNIs were responsible for chronic al-lograft dysfunction and that strategies to minimize CNIsshould be undertaken to preserve allograft function. Thisnow appears to be wrong as several studies demonstratethat ongoing immune events that persist or arise in under-immunosuppressed individuals are likely the most impor-tant cause for graft failure, and this is commonly associatedwith the development of DSAs. Thus, the findings wouldsuggest that DSA-dependent ADCC could also contribute toboth acute and chronic loss of allografts and this might beprevented, at least in part, by maintenance of therapeuticCNI levels. However, the protective role of NK cells viaADCC in viral infection and tumor formation is also es-sential for successful transplantation. Therefore, furtherstudies are required to determine the optimal dose of CNIsand corticosteroids to reduce the incidence of ABMR whileminimizing risks of viral infections.

In conclusion, the potent suppressive effects of CNIsor prednisone on antibody-mediated NK cell activation maycontribute to the reduction of ADCC-mediated ABMR inHS patients and underscore the importance of medicationcompliance and risks of strategies aimed at CNI or cortico-steroid minimization or avoidance in sensitized patients.

MATERIALS AND METHODS

In Vitro Antibody-Mediated NK Cell Activation(In Vitro ADCC)

This study was approved by the Institutional Review Board at Cedars-

Sinai Medical Center. PBMCs were isolated from heparinized blood from

five normal individuals using Ficoll-Hypaque and then mixed (1 � 106

cells) as previously described (7, 8). After the PBMC mixture were irradi-

ated (PBMCx) followed by removal of supernatant by centrifugation at

350g, the cells were mixed with 50 KL of HS sera with panel reactive anti-

body greater than 50% or normal serum with panel reactive antibody 0%

(control), and incubated for 30 minutes at room temperature. After separating

sera by centrifugation at 350g followed by washing with phosphate-buffered

saline, the antibody (Ab)-coated PBMCx were re-suspended in culture me-

dium (RPMI 1640 medium supplemented with 10 mM HEPES, 10% fetal

bovine serum, penicillin, and streptomycin) (1 � 106 cells/mL) and used as

stimulator cells. One hundred microliters of whole blood (responder cells)

from a normal individual was incubated with 100 KL of Ab-coated PBMCx

(stimulator cells), Abs to CD28/49d (1 Kg/mL), and Brefeldin A (10 Kg/mL)

(BD Biosciences, San Jose, CA), with or without various concentrations of

immunosuppressive drugs at 37 -C for 6 hours followed by additional incu-

bation at 18 -C for 12 hours using a standard procedure for the allo-CFC assay

as previously described (7, 8). After incubation, the mixture was submitted for

flow cytometry analysis to measure IFNF production and degranulation in NK

cells. Immunosuppressants were tested at the following concentrations:

CSA (Bedford Lab, Bedford, OH; 200Y5,000 ng/mL), TAC (Selleck Chemicals,

Houston, TX; 25Y1,000 ng/mL), SRL (Selleck Chemicals, Houston, TX;

10Y1,000 ng/mL), MMF (Roche, Nutley, NJ; 100Y50,000 ng/mL), and pred-

nisone (Roxane Lab, Columbus, OH; 500Y100,000 ng/mL).

Measurement of Intracellular IFNF andCD107a Expressed on NK Cells by FlowCytometry Analysis

A similar procedure was used as previously described with minor mod-

ification (7). Briefly, after an 18-hour incubation, 20 KL of EDTA (2mM)

was added to the mixture to stop the reaction and then 20 KL of an Ab

cocktail containing Abs to CD3, CD16, CD56, and CD107a (BD Bio-

sciences) were added followed by incubation at room temperature for

30 minutes. Erythrocytes were lysed with FACS Lysing Solution (BD Bio-

sciences) by incubation at room temperature for 10 minutes followed by

centrifugation at 600g to remove the supernatant. Cells were then perme-

abilized with 500 KL of FACS Permeabilizing Solution 2 (BD Biosciences)

followed by addition of 1 KL of anti-IFNF Ab (BD Biosciences). After cell

acquisition, lymphocytes first gated by forward/side scatter were further

plotted against CD3. Then CD3j cells were plotted against CD56 versus

IFNF or CD107a, and IFNF+ or CD107a+ cell% in CD56+ NK cells was

calculated. Analysis was performed on a BD LSRFortessa with BD FACS

Diva workspace. Results were expressed as the ratio against IFNF+ or

CD107a+ NK cell% in control condition without ISD.

Statistical AnalysisThe effect of each immunosuppressive drug on IFNF production or

degranulation in NK cells (Fig. 2) was assessed by a standard paired two-

tailed t test. A Bonferroni adjustment was used to assess the significance;

P less than 0.0125 for the various concentrations of prednisone and SRL,

and P less than 0.01 for CSA, TAC, and MMF were considered statistically

significant. In the experiments of combination of CNI with prednisone

(Figs. 3 and 4), the effect of CNI or prednisone on IFNF production in NK

cells at each prednisone or CNI concentration, respectively, was first ana-

lyzed by repeated measures ANOVA (RMANOVA). When the RMANOVA

showed P less than 0.05, post hoc analyses were carried out. A Bonferroni

adjustment was used to assess the significance; P less than 0.0167 and P less

than 0.0125 were considered statistically significant for the various con-

centrations of prednisone at each CNI and the various concentrations of

CNIs at each prednisone concentration, respectively. A similar analysis was

performed for the effect of combination of CNI and prednisone on

degranulation.

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