Reversibility with Interleukin-2 Suggests that T Cell Anergy Contributes to Donor-Specific Hyporesponsiveness in Renal Transplant Patients
Wan Fai Ng*,
Maria Hernandez-Fuentes*,
Richard Baker*,
Afzal Chaudhry and
Robert I. Lechler*
*Department of Immunology and Department of Nephrology, Hammersmith Hospital, Imperial College Faculty of Medicine, London, United Kingdom.
Correspondence to Professor Robert I. Lechler, Department of Immunology, Hammersmith Hospital, Imperial College Faculty of Medicine, Du Cane Road, London W12 0NN. Phone: 44-0-20-83832088; Fax: 44-0-20-83832788;E-mail: r.lechler{at}ic.ac.uk
ABSTRACT. Data from various rodent models have implicated arole for anergic T cells in the maintenance of self and transplantationtolerance. The relevance of donor-specific T cell anergy toclinical transplantation, however, has not been demonstrated.Previous studies have reported that recipients of solid organtransplant often have reduced frequencies of CD4+ T cells withanti-donor direct pathway allospecificity after transplantation.The underlying mechanism(s) of this donor-specific hyporesponsivenessis unclear but likely to contribute to the diminished immunosuppressiverequirement of transplant patients with time after transplantation.This study shows that ex vivo treatment of CD4+ T cells fromrenal transplant recipients with IL-2 could specifically increasethe anti-donor frequency in all the patients with evidence ofdonor-specific hyporesponsiveness. It also shows that the IL-2–inducedrecovery of anti-donor frequency is unlikely to result fromnonspecific stimulation or selective clonal expansion of activated,allospecific CD4+ T cells. Taken together, the data suggestthat T cell anergy plays an important role in the direct pathwayhyporesponsiveness that evolves in many human renal transplantrecipients.
Heightened interest in clinical transplant tolerance stems fromthe acquisition of insights into the mechanisms and the adventof novel biologic reagents that may promote such a tolerantstate. For these reasons, it is necessary to understand theevolution of anti-donor responses using current approaches toimmunosuppression. We and others (1–3) have previouslydescribed that a substantial proportion of patients with kidneyor other solid organ transplants have markedly reduced numbersof T cells with direct allospecificity, particularly in long-termlive-related transplant recipients. Although markedly decreasedfrequencies of direct pathway T cells does not equate to tolerance,it is tempting to speculate that mechanisms that contributeto peripheral T cell tolerance may be operative in diminishingthe anti-donor T cell repertoire. It is also important to notethat direct pathway hyporesponsiveness can often be observedin patients with chronic transplant rejection. In these patients,it appears that the indirect pathway of anti-donor alloimmunityprovides an immunologic drive to this indolent process.
Two attractive, yet not mutually exclusive, candidate mechanismsof direct pathway hyporesponsiveness are deletion and anergy.In vitro studies using renal epithelial cells as antigen-presentingcells can result in T cell anergy (4), and peripheral deletionhas been shown to be an important mechanism in maintaining toleranceto foreign antigens (5,6). In this study, we investigated whetherthese mechanisms are operative in the maintenance of donor-specifichyporesponsiveness. Because of the lack of known specific markerof anergic cells, we have relied on the functional propertiesof anergic T cells to distinguish these possibilities. One ofthe characteristics of anergic T cells is that their reactivitycan be restored by cell division driven by added interleukin-2(IL-2) (7). Thus, if the reduction of anti-donor frequency isa consequence of T cell anergy, ex vivo treatment of T cellwith IL-2 may result in restoration of the anti-donor frequency.On the other hand, if deletion is the underlying mechanism ofthe donor-specific hyporesponsiveness, then the anti-donor frequencywill be unchanged with IL-2 treatment.
Patients
All patients in this study were adult recipients of a kidneytransplant at the Hammersmith Hospital at least 6 mo beforethe study. All patients included in this study had functionaltransplants at the time of study. For recipients of cadavericdonor organs, only those with stored donor cells (donor spleen/lymphnode cells or peripheral blood mononuclear cells [PBMC]) werestudied. Relevant clinical details are given in Table 1. Thirdparty controls were chosen, to the extent possible, so thatthe number of mismatches for human leukocyte antigen (HLA)-DRwas the same as that between the donor and recipient. If thedonor and recipient shared a DR allele, a third party stimulatorwas chosen, when possible, that expressed the shared DR allotypebut expressed a different DR alloantigen. This study was approvedby the ethics committee of Hammersmith Hospital.
Cells and Culture Conditions Culture Medium.
In all in vitro assays involving human cells, RPMI-1640 mediumsupplemented with L-glutamine (2 mmol/L; Life Technologies BRL,Paisley, UK), penicillin/streptomycin (100 IU/ml and 100 µg/ml,respectively; Life Technologies BRL), amphotericin (500 ng/ml;Life Technologies BRL), and gentamicin (2 µg/ml; Sigma,Poole, UK), referred to as supplemented RPMI, with 10% humanAB serum (Harlan Sera-Lab, Loughborough, UK) was used. All cellswere incubated at 37°C with 5% CO2 and 95% air.
CTLL-2 Cells.
This murine cell line responds to murine IL-2 and IL-4 but onlyto human IL-2. Cells were maintained in culture in supplementedRPMI medium with 10% fetal calf serum (FCS; BioWhittaker, Wokingham,UK) and 10 U/ml recombinant human (rh)-IL-2 (Boehringer Mannheim,Mannheim, Germany). They were subcultured every 2 to 3 d. Cellswere rested in medium without IL-2 overnight before use in assays.
Responder Cells.
PBMC were isolated from peripheral blood samples from patientsby density gradient centrifugation over Lymphoprep (Nycomed,Birmingham, UK). For isolation of CD4+ T cells, PBMC were incubatedin medium supplemented with 2% FCS at 37°C on tissue culturedishes for 45 min to remove adherent cells. Nonadherent cellswere collected and washed. Non-CD4+ cells were depleted by incubationwith a cocktail of monoclonal antibodies (anti-CD8 [OKT8], anti-CD19[BU12], anti-HLA-DR [L243], collected from supernatants of hybridomas,and anti-CD56 [B159; PharMingen, San Diego, CA]) followed bymagnetic bead (Goat anti-mouse pan-IgG beads; Dynal, Wirral,UK) separation according to manufacturer’s protocol. Thepurity of the cell population was measured by flow cytometryand was always above 90%. The responder cells were then separatedinto two fractions, one being used immediately in limiting dilutionanalysis (LDA). The other fraction was incubated with 30 U/mlrh-IL-2 for 3 d followed by resting in culture medium withoutexogenous IL-2 for 24 h before being used in LDA.
Stimulator Cells.
Spleen cells or lymph node cells were provided by the tissue-typinglaboratory, Hammersmith Hospital. Cells were isolated from smallportions of spleen or lymph node recovered at the time of organretrieval. The spleen was injected with ice-cold RPMI medium,and the cells were washed and separated by density gradientcentrifugation. For live-related donors and third-party controls,peripheral blood samples were obtained, and PBMC were isolatedas above. Cells were then aliquoted and cryopreserved untilrequired. All stimulator cells were gamma-irradiated (30 Gy)before use. In the case of autologous mixed lymphocyte reactionculture, irradiated CD3-depleted PBMC were used as stimulatorcells. PBMC were incubated with supernatant of anti-CD3 mAb(OKT3), followed by magnetic bead (Dynal) separation.
Limiting Dilution Assays
Seven serial dilutions of responder CD4+ cells in 24 replicateswere co-cultured with 5 x 104 irradiated stimulator cells perwell in round-bottomed 96-well plates. For the estimation ofanti-tetanus frequencies, 2 x 104 irradiated autologous CD3-depletedPBMC (pulsed with tetanus toxoid [Evans vaccines, UK], at adilution of 1:1000) were used as stimulator cells. The exactnumber of responder cells added per well depended on the numberof responder cells obtained from each patient, with the topdilutions ranging from 7500 to 80,000 cells per well. After4 d, 100 µl of supernatant from each well was collectedand transferred to another round-bottomed 96-well plate, andIL-2 production was estimated by bioassay using the murine CTLL-2cells. Essentially, 5000 CTLL-2 cells were added to the supernatantand cultured for 48 h, 3H-thymidine was added in the last 12h of the culture. 3H-thymidine incorporation was assessed byliquid scintillation spectrometry. The sensitivity of each assaywas determined separately by measuring the 3H-thymidine incorporationby the CTLL-2 cells at a titration of rh-IL-2 concentrations.Wells were scored positive if the counts were above three SDof the average count of the control (wells containing only irradiatedstimulator cells without responder cells) or the lowest detectionlimit of the assay (calculated from the sensitivity curve),whichever was higher.
Calculation of Precursor Frequencies.
The frequency, confidence interval, and 2 value for each assaywere calculated by the maximum likelihood method using GLIMsoftware (NAG Ltd., Oxford, UK). For all data, a probabilityestimate of the data conforming to "single hit" kinetics wascalculated. Hyporesponsiveness was considered significant ifthe confidence intervals of the anti-donor frequency and theanti–third-party frequency did not overlap and if themeasured anti-donor frequency was less than 1 in 10,000. Changesin frequencies after IL-2 treatment were regarded as significantif the 95% confidence limits of the frequencies before and afterIL-2 treatment did not overlap.
Donor-Specific Hyporesponsiveness Was Detected in 6 of 13 Patients
In agreement with previous studies from our laboratory and others,we found that a significant portion of patients had reducedanti-donor, compared with anti–third-party, CD4+ T cellfrequencies. Thirteen patients were studied, six of which demonstrateddonor-specific hyporesponsiveness (Figure 1). In three otherpatients, the anti-donor CD4+ T cell frequencies were significantlylower than anti–third-party frequencies but higher than1 in 10,000. The maintenance of high anti–third-partyfrequencies argues against this being the result of nonspecificimmunosuppression due to the drug therapy the patients received.Although the anti-donor frequencies pretransplant were not measuredin this study, we have previously reported that reduction inanti-donor CD4+ T cell frequency occurred following transplantation(8).
Figure 1. Precursor frequencies of CD4+ T cells against donor (filled diamonds) and third party (filled squares) antigens before treatment with IL-2.
Treatment of IL-2 Does Not Induce Significant Proliferation of Peripheral Blood CD4+ T Cells
Before investigating the effect of ex vivo treatment of CD4+T cells with IL-2 on the anti-donor frequency, we investigatedwhether IL-2 alone can lead to proliferation of CD4+ T cellsand/or alter the anti–third-party frequency. To excludeany nonspecific effect of IL-2 in enhancing immune responsesof CD4+ T cells, we investigated whether incubating purifiedCD4+ T cells with IL-2 would result in significant proliferationand expansion of the cells, which may affect the result of thesubsequent limiting dilution analyses. CD4+ T cells from peripheralblood of healthy volunteers or renal transplant recipients werepurified and were incubated with 30 units of rh-IL-2. Proliferationwas measured daily. The cells were then washed thrice and culturedfor an additional 3 d without IL-2, and proliferation was againmeasured daily during this rest period. One healthy volunteerand three patients were studied. In all experiments, the CD4+T cells showed minimal proliferation during the 3-d incubationwith IL-2. Upon withdrawal of exogenous IL-2 in the culture,their proliferation returned rapidly to the baseline level after24 h. Figure 2 shows representative data from a normal subjectand a renal transplant recipient.
Figure 2. Proliferative responses of CD4+ cells to IL-2 in (A) a normal subject and (B) a renal transplant recipient. Purified CD4+ cells (1 x 104) were cultured with or without 30 U/ml recombinant human (rh)-IL-2. Proliferation was measured daily. CD4+ cells that had been treated with IL-2 were washed twice before proliferation was measured. After 3 d, the cells were washed and cultured for an additional 3 d without exogenous IL-2, and proliferation was measured daily. The responses to phytohemagglutin (PHA) (2 µg/ml) plus IL-2 (10 U/ml) were used as positive controls and were performed at days 0, 4, and 7.
Treatment with IL-2 Does Not Affect Anti–Third-Party Frequencies as Measured by Limiting Dilution Analysis
Another question concerning the treatment of CD4+ T cells withIL-2 was whether it would affect the anti–third-partyfrequency and make any changes in anti-donor frequency by IL-2treatment difficult to interpret. Three normal subjects andone renal transplant recipient were studied. In all four cases,the third-party frequencies were remarkably similar before andafter IL-2 treatment and their 95% confidence intervals overlapped(Figure 3). Not only did this result exclude nonspecific changesin frequency, but it also indicated that cell viability wasmaintained during the 4 d of culture before performing the limitingdilution analysis.
Figure 3. Anti–third-party frequencies before (filled squares) and after (open squares) IL-2 treatment. N1 to N3 are normal subjects, and P00 is a renal transplant recipient.
IL-2 Specifically Increased the Direct Anti-Donor Frequencies in all Patients who hadDonor-Specific Hyporesponsiveness
Among the six patients in whom donor-specific hyporesponsivenesswas demonstrated, IL-2 significantly increased the frequenciesagainst donor cells in all these patients (Figure 4). In fiveof these patients, the anti-donor frequency was increased tothe extent that the 95% confidence intervals of anti-donor andanti–third-party frequencies overlapped after IL-2 treatment.In the remaining patient, the anti-donor frequency was increasedtenfold. The third party frequencies were unaltered. We usedWilcoxon rank test to determine whether the changes in anti-donorand anti–third-party frequencies were statistically significant.The respective P values for changes in anti-donor and anti–third-partyfrequencies were 0.028 and 0.917, indicating that the changein anti-donor frequencies was statistically significant whilethe third party frequency was not altered by the treatment withIL-2. Furthermore, while the anti-donor frequencies in thisgroup of patients were significantly lower than the anti-thirdparty frequencies before the IL-2 treatment (P = 0.028), thisdifference was obliterated after IL-2 treatment (P = 0.753).In contrast, among the patients in whom no donor-specific hyporesponsivenesswas demonstrated, treatment with IL-2 ex vivo did not increasethe anti-donor frequency (P = 0.398; for patient P5, althoughthe anti-donor frequency was marginally increased after IL-2treatment, the confidence intervals between the anti-third partyand anti-donor frequencies were overlapping before and afterIL-2 treatment). These observations suggest that the reversalof hyporesponsiveness with IL-2 is not a consequence of nonspecificenhancement of immune reactivity but reflects the reversal ofanergy in donor-specific CD4+ T cells.
Figure 4. (A) Anti-donor and anti–third-party frequencies of IL-2-secreting CD4+ T cells before IL-2 treatment ex vivo. (B) Anti-donor and anti–third–party frequencies of IL-2-secreting CD4+ T cells after IL-2 treatment ex vivo were superimposed on Figure 4A to demonstrated the reversal of hyporesponsiveness after IL-2 treatment. Filled diamonds represent the anti-donor frequency before treatment with IL-2; open diamonds represent the anti-donor frequency after IL-2 treatment. Filled and open squares represent anti-third party frequency before and after IL-2 treatment, respectively. The patients have been arranged into groups according to whether they demonstrated donor-specific hyporesponsiveness and reversibility with IL-2.
Specific Enhancement of Anti-Donor CD4+ T Cell Frequency Following Treatment with IL-2 Is Unlikely to Be Due to Clonal Expansion of Activated Donor-Specific CD4+ T Cells
An alternative explanation for the increase in anti-donor frequencyafter IL-2 treatment was the expansion of small numbers of activateddonor-specific T cells. This selective clonal expansion maybe difficult to detect. To examine this possibility, we studiedthe response of a healthy volunteer to tetanus toxoid aftera booster dose of tetanus vaccine. We measured the anti-tetanusfrequency of CD4+ T cells before and after the vaccination andafter treatment of CD4+ T cells with IL-2 ex vivo post-vaccination.The frequency in the absence of added antigen was used as control.After the booster vaccine, we anticipated an increased numberof activated anti-tetanus CD4+ T cells, creating an ideal situationfor the investigation of the responses of activated CD4+ T cellsto the treatment of IL-2. If treatment with IL-2 leads to expansionof activated CD4+ T cells, then we would expect to observe anincrease in anti-tetanus frequency after IL-2 treatment. Asshown in Figure 5, we found that, after vaccination, the anti-tetanusfrequency increased more than 20-fold from 1:280,000 to 1:12,500.However, when the CD4+ T cells were treated with IL-2, the anti-tetanusfrequency was halved. This implies that the donor-specific increasein frequency with IL-2 treatment observed in renal transplantrecipients is unlikely to result from clonal expansion of activatedanti-donor CD4+ T cells.
Figure 5. The anti-tetanus frequency (open circles) of CD4+ T cells of a normal healthy volunteer before booster tetanus vaccine and after vaccination with or without ex vivo treatment with IL-2. The autologous mixed lymphocyte reaction (MLR) frequency (filled triangles) was used as control.
Ever since the demonstration of anergic T cell clones in vitro(9), there has been widespread interest in their role in themaintenance of self and transplantation tolerance. Several studiesusing animal models have provided evidence for a role of anergicT cells in the maintenance of transplant tolerance or prolongationof graft survival (10,11), whether T cell anergy play any rolein the evolution of anti-donor responses in clinical transplantationhas not been examined.
We have previously reported that reduced frequencies of CD4+T cells with direct anti-donor allospecificity was frequentlyobserved after solid organ transplantation. This reduction offrequencies of anti-donor CD4+ T cells was more pronounced inthe CD45RO+ subset of CD4+ T cells. In contrast, the frequenciesof anti–third-party T cells were unaltered after transplantation(1,8). One simple explanation for these findings is that anti-donorT cells are sequestered within the graft, resulting in an apparentloss of these cells in peripheral blood. However, Orosz andBishop (12) used sponge matrix allograft implantation techniqueand limiting dilution analysis to measure the frequencies ofalloantigen-specific T cells at the graft site and in peripheralblood. They found that the frequencies of alloreactive helperT lymphocytes in peripheral blood and at the graft site weresimilar. Thus, the question of why donor-specific hyporesponsivenessis such a common phenomenon in patients with solid organ transplantationremains unanswered. Although markedly decreased frequenciesof direct pathway T cells does not equate to tolerance, it istempting to speculate that mechanisms that contribute to peripheralT cell tolerance may be operative in diminishing the anti-donorT cell repertoire.
In this study, we have demonstrated that donor-specific hyporesponsivenesscan be specifically reversed by ex vivo treatment of recipientCD4+ T cells with IL-2 in all patients, consistent with thehypothesis that anergy contributed to the decrease in anti-donorfrequencies. The third-party frequencies were unaffected. Theconsistency of the anti–third-party frequencies not onlydemonstrated the reproducibility of the assays but also indicatedthat the increase in anti-donor frequency after IL-2 is unlikelyto be due to nonspecific stimulatory effect of IL-2. In thisstudy, the cell number after IL-2 treatment of the CD4+ cellswas usually reduced rather than increased. In addition, thepoor proliferative response to IL-2 of the CD4+ T cells arguesagainst such an explanation. Moreover, the ability of IL-2 toincrease the anti-donor frequency was restricted only to patientswho exhibited donor-specific hyporesponsiveness. Nevertheless,it remains possible that IL-2 treatment had expanded the numbersof direct allospecific T cells rather than reversed anergy.However, our observation that IL-2 treatment in vitro led toa decrease in anti-tetanus frequency in a volunteer who hada recent booster vaccine argues against such an explanation.This reduction in frequency may be a result of activation inducedcell death in response to IL-2 (13,14).
It is interesting that in the three patients in whom the anti-donorCD4+ T cell frequencies were above 1 in 10,000 but neverthelesssignificantly reduced to anti-third party frequencies, IL-2treatment failed to increase the anti-donor frequencies. Oneexplanation is that other mechanism(s) is/are operative; forexample, deletion and/or regulation. We have recently shownthat a naturally occurring subpopulation of CD4+ T cells, characterizedby the constitutive expression of CD25, has the ability to regulatealloresponses (15). Interestingly, unlike anergic T cells, CD4+CD25+cells do not proliferate in response to exogenous IL-2, andthey remain hyporesponsive and retain their regulatory functionafter exogenous IL-2 is withdrawn. Second, it has been suggestedthat there are different levels of T cell unresponsiveness thatare associated with different degrees of reversibility (16).Finally, it has been reported that cyclosporin may prevent theinduction of anergy (17,18). However, the effect of cyclosporinon anergy induction is far from clear; many studies have reportedthe induction of anergy in the presence of cyclosporin (19–23).The reason for the discrepancies between different studies isunclear but may be related to the method of anergy inductionand the dose of cyclosporin used. In this study, we did notfind any correlation between the use of cyclosporin and thereversibility of donor-specific hyporesponsiveness with IL-2.
Another interesting question is the lifespan of anergic cells.In vitro data suggest that anergic T cell clones can be maintainedfor weeks (7). Our data suggest that either anergic cells havea long lifespan, or, alternatively, a dynamic process of encounterof recipient T cells with donor MHC molecules is in operationto maintain a cohort of anergic direct pathway T cells.
The finding that IL-2–driven cell division can reversehyporesponsiveness in direct pathway T cells may have relevanceto the link between systemic infections such as CMV or localinfections of the urinary tract and acute rejection episodes.Conceivably, infection of the urinary tract can result in productionof IL-2 locally or in the draining lymph nodes, leading to reversalof the anergic state of the allospecific T cells and consequentacute rejection. Theoretically, anergy reversal of allospecificT cells could also contribute to the process of chronic rejection;however, there was no correlation between reversible hyporesponsivenessand chronic rejection in our study. The observation that chronicrejection occurs despite hyporesponsiveness in the direct pathwayT cells indicates that an alternative mechanism, such as theindirect pathway of the anti-donor T cell response, may playa key role in orchestrating the chronic rejection process. Thus,it is important to devise strategies to induce tolerance toboth the direct and indirect allospecific pathway if clinicaltolerance is to be achieved.
In conclusion, our findings are consistent with the existenceof anergic direct pathway T cells in a proportion of renal transplantpatients and suggest that anergy may be an important contributorymechanism to the maintenance of donor-specific hyporesponsivenessin clinical transplantation.
Acknowledgments
We are grateful for Drs. A. George and G. Lombardi for theirhelpful comments on the manuscript. We also thank Janet Watersfor coordinating the collection of blood samples, N. Olsen-Saraiva-Camarafor assistance in statistical analysis, and the staff of tissue-typinglaboratory for analyzing third-party volunteers’ tissuetypes. WFN is a recipient of a Wellcome Trust Fellowship.
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Received for publication May 9, 2002.
Accepted for publication August 1, 2002.
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Abstract
Introduction
2 value for each assay were calculated by the maximum likelihood method using GLIM software (NAG Ltd., Oxford, UK). For all data, a probability estimate of the data conforming to "single hit" kinetics was calculated. Hyporesponsiveness was considered significant if the confidence intervals of the anti-donor frequency and the anti–third-party frequency did not overlap and if the measured anti-donor frequency was less than 1 in 10,000. Changes in frequencies after IL-2 treatment were regarded as significant if the 95% confidence limits of the frequencies before and after IL-2 treatment did not overlap. 
