Transplant Immunology 13(2004) 55–61

0966-3274/04/$ - see front matter� 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.trim.2004.04.003

Ex vivo lymphocyte proliferative function is severely inhibited in renaltransplant patients on mycophenolate mofetil treatment

Paul Hutchinson *, Matthew Jose , Robert C. Atkins , Stephen R. Holdswortha, b c d

Department of Medicine, Monash University Monash Medical Centre, 246 Clayton Road, Clayton, VIC 3168, Australiaa

Department of Nephrology Monash Medical Centre, 246 Clayton Road, Clayton, VIC 3168, Australiab

Department of Nephrology Monash Medical Centre, 246 Clayton Road, Clayton, VIC 3168, Australiac

Department of Medicine, Monash University Monash Medical Centre, 246 Clayton Road, Clayton, VIC 3168, Australiad

Received 21 January 2004; received in revised form 13 April 2004; accepted 16 April 2004

Abstract

Mycophenolate mofetil(MMF) is a recently introduced immunosuppressive drug. Its active form is mycophenolic acid(MPA).MPA specifically inhibits de novo purine synthesis, which is vital for T and B lymphocyte proliferation. We measured lymphocytesubset numbers and mitogen induced proliferation in kidney transplant recipients on different combinations of MMF, cyclosporinA (C), azathioprine(A) and prednisolone(P) (CqA ns70; CqAqP ns15; CqMMF ns45; CqMMFqP ns37) andnormals (ns73). Patients on MMF had severely reduced phytohaemagglutinin A(PHA) induced proliferation compared tonormals (Nml 2766"926 CPMy1000 lymphocyteswmean"S.D.x; CqMMF 282"406; CqMMFqP 195"496); non-MMFpatients did not differ from normal. Similar inhibition of Poke Weed Mitogen and Staphylococcal enterotoxin B inducedproliferation was observed. Cell cycle studies established that MMF patients had a significantly higher proportion of lymphocytesin the G0yG1 phase following PHA stimulation than the non-MMF patients. All transplant groups had significantly lower B cellnumbers than the normal controls but no differences in CD4 and CD8 T cell numbers. All but the CqMMF group hadsignificantly lower CD16qNK cell numbers than normal, while only the non-MMF groups had significantly lower CD56qNKcell numbers. The proliferation assay used was an ex vivo diluted whole blood technique. Removal of residual MPA by washingthe plasma out prior to mitogen stimulation led to a significant increase in proliferation in six out of seven cases. In summary wehave found that MMF treatment has a strikingly inhibitory effect on patient ex vivo lymphocyte mitogenic function.� 2004 Elsevier B.V. All rights reserved.

Keywords: Mycophenolate mofetil; Transplantation; Lymphocytes; Proliferation

1. Introduction

Mycophenolate mofetil(MMF) is a recently intro-duced immunosuppressive agent for organ transplanta-tion w1x. In vivo this drug is deesterfied into its activeform mycophenolic acid(MPA). MPA is a potent andspecific inhibitor of inosine 59-monophosphate dehydro-genase(IMPDH), a rate-limiting enzyme involved inthe de novo synthesis of purines. Unlike over cell typesthat rely on the salvage pathway of purine synthesis, Tand B lymphocyte proliferation is dependent on the de

*Corresponding author. Tel.:q61-3-9594 3565; fax:q61-3-95943611.

E-mail address:Paul.Hutchinson@med.monash.edu.au(P. Hutchinson).

novo purine pathway. In vitro studies have shown thatMPA blocks T and B cell proliferation in the transitionfrom G1 into the S phase of the cell cycle, but does notinhibit early events in lymphocyte activation such ascytokine production w2x. Because MMF specificallyinhibits T and B lymphocyte proliferation it appears anideal immunosuppressive agent in blocking acute rejec-tion following organ transplantation.

Initial studies on transplants of a hearts in micew3xand kidneys in dogsw4x found that MMF preventedacute allograft rejection and appeared to have few toxicside effects. In humans, a multi-centre trials of renalallograft patients has found that treatment with MMFleads to reduced rates of rejection of up to 50%compared to non-MMF treated patientsw5x.

56 P. Hutchinson et al. / Transplant Immunology 13 (2004) 55–61

The success of the currently used immunosuppressiveagents such as MMF has led to longer graft survival forkidney transplant patients, which in turn has meant thatpatients are now being exposed to immunosuppressivedrugs for longer. Their effects on the immune systemsof these patients may be responsible for the higher ratesof infection w6x and cancerw7x amongst this population.An important function of the cognate immune system isthe ability of T and B cells to respond to non-selfantigen by clonal proliferation. The measurement of themitogenic lymphocyte response is a well-accepted assayfor modelling this function. Using an ex vivo wholeblood stimulation technique we assessed the mitogeniclymphocyte proliferation response of a group of renalallograft recipients. The effects of various immunosup-pressive regimes were compared. Patients on MMF hada significantly inhibited mitogen induced proliferationcompared to those patients not on MMF and normals.

2. Objective

Assessment of the effects of mycophenolate mofetilon renal transplant patient lymphocyte mitogenic func-tion and subset numbers as compared to non-MMFtreated renal transplant patients and normal controls.

3. Materials and methods

3.1. Patient and normal groups

The patient group studied consisted of 167 adult renaltransplant recipients. Inclusion criteria were:)6 monthstransplant time, serum creatinine-250 mmolyl, and noconcomitant clinical condition or therapy(other thanroutine immunosuppression) known to affect immunefunction. Normals(ns73) consisted of laboratory staffand others, who were under no medication and had nomedical problems at the time of assessment.

Total white cell counts were obtained from full bloodevaluations(Abbott Diagnostics Cell-Dyn cell counter,Abbott Park, IL) by Monash Medical Centres pathologyprovider Southern Cross Pathology. Immunoglobulinsubclass levels, serum creatinine, and blood cyclosporintrough concentration were also performed at SouthernCross Pathology(Dade Dimension RXL autoanalyser,Dade Behring, Deerfield, IL).

3.2. Immunosuppressive protocols

Over the 5 years of data acquisition several combi-nations of four standard immunosuppressive agents wereemployed including azathioprine(AZA) 1–2 mgykgyday, mycophenolate mofetil 1–2 gyday (both adjustedto maintain WCC)4500yml), cyclosporine A(mainte-nance dose 3–6 mgykgyday), and prednisolone initially20 mgyday until 3 months then tapered and withdrawn

over 7 months or continued as maintenance therapy inselected patients(5–10 mgyday). Four different main-tenance drug regimes were employed; cyclosporine Aplus azathioprine(CqA, ns70), cyclosporine A, aza-thioprine plus prednisolone(CqAqP, ns15), cyclo-sporine A plus mycophenolate mofetil(CqMMF, ns45), cyclosporine plus mycophenolate mofetil plusprednisolone(CqMMFqP, ns37). Rejection episodeswere treated with pulse IV methyl prednisolone 0.5–1gyday for 3 days and refractory rejection with OKT3(ns15) for 7–10 days. Fourteen patients were switchedfrom azathioprine to mycophenolate mofetil during thisstudy. This was due to the policy of the unit and didnot result in any other changes to the therapy the patientswere receiving. Four other patients were switched backto azathioprine due to mycophenolate mofetil inducedgastro-intestinal intolerance.

3.3. Whole blood culture and mitogen stimulation

Blood was collected into tubes containing LithiumHeparin anticoagulant. For the proliferation assays, theblood was diluted 1y10 with tissue culture medium(RPMI 1640, ICN Biomedicals) supplemented with 50Uyml penicillin (ICN Biomedicals) and 50 mgymlstreptomycin(ICN Biomedicals) and 200ml cultured intriplicate wells of a 96 well plate(Disposable Products,Australia) with a range of doses(0–5 mgyml) ofPhytohaemagglutinin A(PHA, Wellcome, UK), Staph-ylococcal enterotoxin B(SEB, Sigma, Australia), orPoke Weed Mitogen(PWM, Sigma, Australia). Allcultures were maintained at 378C and 5% CO . For the2

cell cycle measurements 2 ml of the 1y10 blood dilutionwas cultured with 5mgyml PHA for 72 h.

To remove plasma from samples prior to mitogenstimulation 1 ml of blood was washed twice in 10 mlof tissue culture medium and then resuspended in tissueculture medium plus 5% fetal calf serum(CSL, Austra-lia) in an equivalent volume to the whole blood assay.

3.4. w Hx Thymidine incorporation3

Mitogen induced proliferation was measured after 72h by addition of 0.5mCi of w Hxthymidine (Amersham3

International, UK) per well over the last 4 h of culture.The cells were harvested onto glass fibre disks(Cam-bridge Technology, MA) using a PHD multi-well cellharvester(Cambridge Technology). The disks were leftto dry overnight before the addition of scintillation fluid(Optiscint Hisafe, Wallac, Finland). Incorporatedw Hx-3

thymidine was assessed using a LKB Wallac beta-counter(Wallac). The counts per minute obtained foreach triplicate dose was averaged and ratioed over thestarting lymphocyte count and expressed as CPMy1000lymphocytes. The peak response was reported as thelymphocyte mitogen response value.

57P. Hutchinson et al. / Transplant Immunology 13 (2004) 55–61

Fig. 1. PHA induced proliferation for normals and four treatmentgroups of allograft recipients. Mean and standard deviation is shown.*P-0.001 vs. Nml.

3.5. Cell surface immunofluorescent staining

Lymphocyte surface antigen expression was assessedwith three colour immunofluorescent labelling. 100mlof blood was incubated at room temperature for 30 minwith combinations of the following antibodies: CD2-phycoerythrin(PE) (Exalpha, Boston, MA), CD3-Cya-nine5 (CY5) (Cymbus, UK), CD4-PE(Cymbus, UK),CD8-Alexa 488(OKT8, home brew), CD14-Alexa 488(90.3.8w8x, in house preparation), CD16-fluroscein iso-thiocyanate (FITC) (Dako, Denmark), CD19-FITC(Cymbus, UK), CD25-PE(Beckman-Coulter, Hialeah,FL), CD45-CY5 (71.5.57, w8x, in house preparation),CD56-PE (Exalpha, Boston, MA). At the end of theincubation period the red cells were lysed using theCoulter Q-prep system(Beckman-Coulter, Hialeah, FL)on the 35-s cycle.

3.6. Cell cycle staining

After the three days of culture the red blood cellswere lysed in an ammonium chloride solution and theremaining leukocytes washed once in PBSy1% FCSy0.01% Azide. Following this the cells were resuspendedin 0.5 ml of PBS and then added to 4.5 ml of ice cold70% ethanol. The cells were left overnight at 48C, orat y20 8C for 2 h, and then spun down and washedonce in PBS. The cell pellet was then resuspended in apropidium iodide(PI) staining solution consisting of 20mgyml PI and 200mgyml RNAse in 0.2% Tween 20.The cells were left at 378C for 15 min and thenanalysed by flow cytometry.

3.7. Flow cytometry

The labelled cells were measured on a Mo-Flo flowcytometer(Dako-Cytomation, Fort Collins, CO), using488 nm(argon laser) and 633 nm(HeNe laser) excita-tion and collecting forward and right angle light scatteras well as FITC(530"15 nm), PE (570"15 nm), andCY5 or PI (670"20 nm) fluorescence. These data wereanalysed using Summit software(Dako-Cytomation),with a gate being placed around the lymphocyte popu-lation in the forward vs. right angle histogram and theirfluorescence signals measured for at least 3000 countsfor the immunofluorescence and 10 000 counts for cellcycle staining.

3.8. Statistics

ANNOVA analysis of the normal and patient treat-ment group results to determine statistically significantdifferences was used. Significance was defined asP-0.05. The analysis was executed by the Graph Pad Prismsoftware program(Graph Pad Software, San Diego,CA).

4. Results

4.1. PHA Stimulated lymphocyte proliferation

Taken as a whole, the transplant population had asignificantly reduced level of proliferation in responseto the mitogen PHA compared to normals:(patients:1223"1372 { mean"S.D.} CPMy1000 lymph., nor-mals: 2766"917,P-0.001). Treatment regimes includ-ing MMF were associated with a marked reduction inmitogen induced lymphocyte proliferation(CqMMFand CqMMFqP P-0.001 vs. Nml) whereas those notincluding MMF had no significant effect(Fig. 1). Therewas no significant difference in the doses ofcyclosporineA and prednisolone prescribed to the patientgroups. The patient groups had similar demographicsexcept that transplant duration time of patients on MMFwas lower than those receiving azathioprine(Table 1).This was expected as MMF was the most recentlyintroduced immunosuppressive drug used by thesepatients.

The effect of switching from AZA to MMF onlymphocyte proliferation was studied in 18 patients. Inall of the patients it was found that switching to MMFtreatment led to a decrease in PHA induced lymphocyteproliferation(mean"S.D.: 81%"17%) (Fig. 2).

4.2. Effect of MMF on lymphocyte subset numbers

Lymphocyte subset numbers and proportions wereevaluated in each group of allograft recipients andcompared to a group of normals. It was found that there

58 P. Hutchinson et al. / Transplant Immunology 13 (2004) 55–61

Table 1Patient demographics

CqA CqAqP CqM CqMqP

Age (years) 47"13 45"12 48"11 47"12Duration of transplant(years) 7.6"3.3* ˆ 5.5"3.1* 5.0"3.7* 2.2"2.2Maleyfemale 38My32F 6My9F 31My14F 23My14FOKT3 Use 4y70 4y15 5y45 2y37

The patient demographics for each of the treatment groups.*P-0.01 vs. CqMqP, P-0.001 vs. CqM.ˆ

Fig. 2. Averaged PHA induced proliferation shown for individualpatients when on a treatment regime with or without MMF.

Fig. 3. B cell numbers for normals and four treatment groups of allo-graft recipients. Mean and standard deviation is shown. *P-0.001 vs.Nml and P-0.05 vs. CqMMF; P-0.001 vs. Nml.ˆ

were no significant differences in CD4 and CD8 T cellnumbers amongst the treatment groups and normalcontrols. Significantly lower B cell counts were foundin all transplant groups when compared to normal whilethe CqMMF group had significantly higher numbers ofB cells when compared to the non-MMF treatmentgroups(P-0.05, Fig. 3). All but the CqMMF grouphad significantly reduced CD16q NK cell numbers(Nmls 253"163yml; CqA 144"226yml, P-0.001;CqAqP 174"242yml, P-0.05; CqMMF 225"129yml; CqMMFqP 153"104yml, P-0.05), while onlythe non-MMF groups had significantly reduced CD56qNK cell numbers (Nmls 235"155yml; CqA121"208yml, P-0.001; CqAqP 155"239yml,P<0.05; CqMMF 211"124yml; CqMMFqP152"95yml).

No significant differences were found between thedifferent treatment groups and normals for the numberof T cells expressing the activation marker CD25.However, all transplant treatment groups had higher thannormal numbers of T cells expressing MHC Class II(P-0.01) but no significant difference was foundbetween treatment groups: Nmls 115"81yul, CqA

262"206yul, CqAqP 305"230yul, CqMMF352"300yul, CqMMFqP 344"316yul.

4.3. Effect of MMF on other mitogen inducedproliferation

In a limited group of patients the effects of MMFtreatment on other lymphocyte mitogens was studied.For both Poke Weed Mitogen(PWM) and Staphylococ-cal enterotoxin B(SEB) the inclusion of MMF in thetreatment protocols was associated with a significantinhibition of lymphocyte proliferation compared to non-MMF treated patients: non-MMF Patients SEB1354"440 CPMy1000 lymphs (ns4), PWM1204"1164(ns4); MMF Patients SEB 221"334(ns7, P-0.001), PWM 121"173 (ns6, P-0.05).

4.4. Washing out plasma reduces the MMF effect onmitogen induced proliferation

To determine if the inhibitory effect of MMF treat-ment on mitogen induced proliferation was due toresidual MPA in the plasma washed cells were comparedwith the whole blood assay. Blood from each patientwas split into two aliquots. One was setup as the usualdiluted whole blood assay while the other was washedtwice with tissue culture medium to remove the plasmaand then the cells resuspended in RPMIy5% FCS andstimulated with mitogen. It was found that in six out of

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seven patients on MMF the washed cells had anincreased proliferative response to PHA compared to thenon-washed cells(non-washed 282"466 CPMy1000lymph; washed 1117"509). In contrast four out of sixnon-MMF patients tested had a decreased proliferativeresponse following washing(non-washed 2531"1218;washed 1617"822).

4.5. Effect of MMF on lymphocyte cell cycle statusfollowing mitogen stimulation

To determine the effect MMF on cell cycle statustotal cellular DNA content was assessed on patientswith and without MMF treatment after PHA stimulationfor 3 days. It was found that those patients receivingMMF had a significantly higher proportion of lympho-cytes in the G0yG1 phase(yMMF 78"5% qMMF90"7%, P-0.01).

5. Discussion

Mycophenolate mofetil(MMF) is a relatively newimmunosuppressive drug that is now being widely usedin the treatment of renal transplant patients. Its mainimmunosuppressive effect is derived from its ability tospecifically inhibit the de novo purine synthesis pathway,which is vital for proliferation of T and B lymphocytes.This effect is associated with reduced rejection withinitial studies demonstrating a 50% reduction in theincidence of acute rejection in patients on MMFw9x.

Our results show that MMF treated kidney transplantpatients have significant inhibition of their mitogeninduced T and B lymphocyte proliferation as measuredex vivo in a diluted whole blood assay; both whencompared to a normal population and against a non-MMF treated renal transplant groups. This reduced levelof mitogen proliferation in the MMF patient groups wasnot due to higher prescribed doses of non-MMF drugs.The MMF patients studied did have a shorter transplantduration time than the non-MMF groups but this isunlikely to account for the inhibitory effect since thelonger duration of immunosuppressive treatment is gen-erally associated with greater immunosuppression. Mito-gens directed against T cells(PHA and SEB) and Bcells (PWM) were both found to have reduced prolif-erative effects in MMF treated patients. To confirm thatthe diminished thymidine incorporation in MMF patientswas due to inhibited proliferation we measured the cellcycle status of the mitogen stimulated cells. It was foundthat the MMF treated patients had a significantly higherproportion of cells in the G0yG1 phase compared tonon-MMF patients, indicating reduced proliferation. Asothers have indicatedw10x this effect seems to be duein a large part to residual MPA(the metabolised formof MMF) in the patient plasma as removal of the plasma

by washing increased the proliferation above that seenwith the unwashed samples.

Previous studies investigating the effects of MMF onkidney transplant patient lymphocyte proliferative func-tion have shown varying results. In van Beusow et al.w11x T cell recall antigen and mixed lymphocyte cultureproliferative responses were assessed in patients treatedwith either AZA or MMF. It was found that there wereno significant differences between the two groups. Inanother study it was observed that MMF treated liver orkidney transplant patients had a substantial decrease inproliferation in the mixed lymphocyte response althoughno difference was detected between MMF treatedpatients and non-MMF patients after PHA stimulationw12x. In both studies the investigators isolated the patientmononuclear cells before stimulation and so residualMPA would have been removed. Rats treated with MMFwere found to have significantly reduced proliferativeresponses to the mitogen Concanavalin A and to anti-CD28 plus Phorbol Myristate Acetate when measuredin a diluted whole blood assay employing both tritiumlabelled thymidine incorporation and cell cycle status12 h after the animals were treated with a single doseof MMF w13x. Stalder et al.w14x found a small groupof renal transplant patients on cyclosporine, prednisoneand MMF had significantly reduced proliferativeresponse to Con A when compared to a group of normalsin a diluted whole blood assay. However, unlike thecurrent study they did not compare the response to non-MMF treated transplant recipients and only assessed alimited number of patients(ns8).

Despite the measured inhibition of mitogen inducedproliferation MMF treatment had little effect on periph-eral blood lymphocyte subset numbers compared to non-MMF treatment patients, with the main difference beingthe higher NK cell numbers in the MMF patients. Asimilar increase in B cell numbers in CqMMF treatedpatients compared to CqA patients has been observedby Francois et al.w15x. The same study found that MMFpatients also had significantly higher numbers of CD8cells. This appears to differ from the results of thecurrent study but could be accounted for if the CD8cells were not co-stained with CD3. In this case, CD8positive NK cells would be included in the calculationfor total CD8 cells. In the current study NK cell numberstended to be higher in the MMF patients compared tothe non-MMF patients. This would explain the increasein CD8 positive lymphocytes observed by these inves-tigators. Other studies have not found a difference inlymphocyte subset populations between MMF and non-MMF immunosuppressive treatment protocols followingkidney allograft transplantationw16x.

How this ex vivo inhibition of T and B cell prolifer-ation by MMF maybe reflected in vivo is unclear. Anti-viral immune responses often lead to expandedpopulations of specific cytotoxic T cellsw17x, indicative

60 P. Hutchinson et al. / Transplant Immunology 13 (2004) 55–61

of in vivo proliferation. Inhibition of this proliferationmay impact on anti-viral responses of the host. Reportson the effect of MMF on viral illnesses are conflicting.The Tricontinental study groupw5x found that patientson the highest MMF dose studied(1.5 g twice daily)did have a greater incidence of cytomegalovirus(CMV)disease and associated tissue invasion compared topatients on AZA. This effect was not observed inpatients treated with a lower dose(2 g MMFyday) ofMMF. It was also found that there were no differencesbetween the groups in the incidence of herpes simplexor zoster at this or the higher MMF dose. More recentstudies, however, have found that treatment with MMFis associated with an increased incidence of CMVinfection w18x and duration of CMV infectionw19x.Another group observed an association between highmycophenolic acid plasma concentrations and severepresentation of herpes virus infections in four patientstreated with MMFw20x.

Since the generation of antibody against antigenrequires B-cell proliferation the ability of vaccines toelicit antibody responses is another parameter of immunefunction in which the observed effects of MMF onmitogen proliferation might be measured. In one studya normal group and two groups of renal allograftrecipients, one treated with PqAqC and the other PqMMFqC, were immunized with an influenza vaccinew21x. It was found that the patients treated with MMFhad a markedly reduced antibody response to the vaccinecompared to the non-MMF patients and the normals.Another group found that when renal transplant patientson MMF were immunized with keyhole limpet hemo-cyanin the IgG response to this antigen was significantlylower than transplant patients not on MMFw22x. Thesame study also found that when MMF patients werere-immunized with tetanus toxoid their induced IgGresponse was also significantly reduced compared tonon-MMF patients. Interestingly, MMF treatment wasnot associated with any reduction in T cell proliferationagainst these antigens.

In summary, transplant recipients had their mitogeninduced lymphocyte proliferation tested using an exvivo whole blood assay. It was found that patientsreceiving MMF as part of their immunosuppressivetherapy had a significant and striking reduction in theirmeasured proliferation compared to those patients notreceiving MMF. What effect this may have on in vivoimmune function remains to be determined.

Acknowledgments

The authors would like to thank Lucy Gathercolefrom the Department of Clinical Immunology, MonashMedical Centre for help with patient assays.

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