(Circulation. 1995;92:862-874.)
© 1995 American Heart Association, Inc.
Articles |
Frequency of Hypoxanthine Guanine Phosphoribosyltransferase (HPRT-) T Cells in the Peripheral Blood of Cardiac Transplant Recipients
A Noninvasive Technique for the Diagnosis of Allograft Rejection
From the Department of Pathology (A.A.A., A.M., J.B.S., M.B.G., K.W.S., F.V.) and the Division of Cardiothoracic Surgery, Department of Surgery (K.K.), Emory University School of Medicine, Atlanta, Ga, and the Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Md (C.O.S., A.H.).
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Histological evaluation of serial endomyocardial biopsies performed at fixed time intervals after cardiac transplantation is the universal method used for the detection of cardiac rejection and assessment of the adequacy of antirejection therapy. No noninvasive methodology thus far investigated has achieved a high enough sensitivity and predictive accuracy to be considered as a potential replacement for endomyocardial biopsy in the detection of rejection in adults. The present study exploited the finding that the rate of spontaneous mutation in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene is higher in proliferating human T cells than in resting cells. Thus, it was reasoned that in the posttransplantation setting, the frequency of HPRT- cells in peripheral blood may provide an indirect measure of alloactivated T lymphocytes.
Methods and Results This study consisted of determining the
clonal frequency of HPRT- mutant cells
(FMC/106 peripheral blood mononuclear cells
[PBMCs]) within a total of 293 peripheral blood samples
representing various numbers of sequential samples from each of
27 transplant recipients. These sequential samples
represented time periods when endomyocardial biopsy
specimens showed either (1) no evidence of rejection (n=5 patients),
(2) a single initial episode after transplantation of early (<1 year)
or late (>1 year) rejection (n=12 patients), or (3) multiple
rejection
episodes (n=10 patients). Statistical analyses were used to quantify
the time profiles of FMC/106 PBMCs in serial samples
among transplant recipients and to determine the association of these
profiles with both the onset of first rejection episodes and, in
appropriate patients, the recurrence of rejection episodes. Data showed
that PBMCs from patients with no evidence of rejection uniformly gave
low values of <6 FMC/106 cells, a frequency similar
to that seen in healthy nontransplanted volunteers. In contrast, 19 of
the 22 PBMC samples that were obtained from patients whose
corresponding biopsy sample was diagnosed with a histological rejection
grade of 
3 gave values of >6 FMC/106 cells, 11 of
which gave values >50/106 cells (range, 146 to
46 982 FMC/106 cells). A significant association
between the onset of first rejection and an increased rate of
FMC/106 values was noted (P=.0001). The
ability of a rising trend in FMC/106 values to
correctly identify the onset of rejection was 81.8% and to correctly
identify no rejection, 100%. In addition, a significant association
between recurrent rejection episodes and persistence of high
FMC/106 values in the weeks after treated rejection
episodes was noted (P=.0003). The ability of a persistently
elevated trend in values of FMC/106 cells to
correctly identify recurrent rejection was 90% and to correctly
identify no rejection, 100%.
Conclusions Increasing frequencies of HPRT- mutant cells in peripheral blood correlated with the onset of first rejection, and persistently elevated HPRT- mutant cells in the weeks after a treated rejection episode correlated with recurrent rejection. This quantitative noninvasive assay may thus serve as a useful adjunct to endomyocardial biopsy for monitoring post–cardiac transplantation patients, and its use as a prospective diagnostic tool merits further study.
Key Words: transplantation • rejection • lymphocytes
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Histological grading of endomyocardial biopsies (EMBs) of donor heart tissue after transplantation has been and continues to be used for the diagnosis of rejection and for the institution of immunosuppressive drug regimens. Although this technique is relatively safe, it has several drawbacks, including sampling error and variation in criteria used for grading, and it is not without risk to the patient.1 In efforts to arrive at a more refined method for diagnosing rejection, numerous attempts have been made to develop noninvasive techniques and/or supplemental studies of aliquots of biopsy specimens. The noninvasive techniques are predicated on either the detection of a physiological abnormality of the rejecting heart2 3 4 or the detection of a variety of immunologic parameters that have been implicated in allograft rejection. The noninvasive techniques that have been studied to date have recently been summarized.5 6 Studies of EMB specimens have included phenotypic characterization of mononuclear cell infiltrates,7 8 9 10 expression of major histocompatibility complex (MHC) class I/II antigens,11 12 cell adhesion molecules,13 14 15 16 17 detection and/or quantification of mRNA coding for cytokines,14 18 19 extracellular matrix proteins,20 21 and studies of the function, specificity, Vß TcR gene usage, and donor-specific committed and precursor frequency analysis of mononuclear cells cultured from such biopsies.7 22 23 24 25 26 27 28 More recently, the finding of a high degree of correlation between graft rejection and increased frequencies of in vitro cultured graft infiltrating cells that are anti-CD8 monoclonal antibody–resistant (high-affinity) donor-specific cytotoxic T lymphocytes (CTLs) in EMB specimens has added another dimension to the diagnosis of allograft rejection.29 These latter techniques, however, still require the procurement of biopsy specimens. Unfortunately, none of the noninvasive techniques used have provided sensitive and reproducible data that can be used solely as an adjunct to studies of EMB-based diagnosis.
The hypoxanthine guanine phosphoribosyltransferase (HPRT) gene encodes for an enzyme that is required by mammalian cells to synthesize DNA via the salvage pathway. This gene, for reasons that are unclear at present, is hypersensitive to spontaneous mutations at multiple regions of the sequence. If a T cell sustains a mutation in this gene that either reduces or abolishes HPRT enzyme activity, the cell has to utilize the much less energy-efficient de novo pathway of DNA synthesis. Thus, culturing of T cells in the presence of the purine analogue 6-thioguanine (6-TG) leads to death of cells that are HPRT+, allowing the selective enrichment of T cells that are HPRT- that survive in media containing 6-TG. These facts have been used successfully by the laboratories of Albertini et al,30 Allegretta et al,31 and Gmelig-Meyling et al,32 who have demonstrated that, upon in vitro culture, T cells from patients with multiple sclerosis and from patients with systemic lupus erythematosus undergoing in vivo myelin basic protein (MBP)–specific and/or generalized activation and proliferation, respectively, have markedly enhanced frequencies of HPRT- mutant cells compared with frequencies shown by healthy subjects. This enrichment of HPRT- actively proliferating cells is reasoned to be secondary to the fact that these activated cells, according to logic, have a higher chance of undergoing random spontaneous mutation for the HPRT- gene than do resting cells. Thus, measurement of mutations in the HPRT gene will permit the estimation of the in vivo frequency of mutant T cells (FMC) as a crude indicator of ongoing and/or past T-cell proliferation. It is reasoned that during rejection episodes, there are likely to be considerable host anti-graft T-cell responses, leading to cell activation and release of cytokines that potentially activate nonspecific bystander peripheral blood cells in situ. A fraction of these activated cells may leave the graft and recirculate. By comparing frequencies of T cells grown in the absence and presence of 6-TG, it may be possible not only to measure the frequency of HPRT- mutant cells (hence the degree of activation) but also to permit enrichment for clones with donor specificity involved in the rejection response. Data derived from a total of 293 peripheral blood mononuclear cell (PBMC) samples obtained in consecutive batches from each of 27 patients at various times after cardiac transplantation constitute the basis of this report. Results indicate that increased frequencies of mutant cells correlate with rejection episodes. More importantly, results of longitudinal studies of the included patients showed that an increase in FMC is predictive of an upcoming rejection episode. Finally, our results document the value of this technique for identifying patients who are at risk for recurrent rejection after being treated for a rejection episode. In concert with EMB and other clinical findings, this assay may be a useful diagnostic tool for monitoring post–cardiac transplantation patients.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Patients
A total of 27 adult patients undergoing cardiac transplantation at Emory University Hospital were the source of peripheral blood samples that were analyzed retrospectively in detail. Samples for analysis were chosen so that they would constitute a series of consecutive samples from patients who experienced no rejection episode (n=5), patients who experienced a single rejection episode early after transplantation (arbitrarily chosen as <1 year, n=6), patients who experienced a single rejection episode late after transplantation (>1 year, n=6), and patients who experienced multiple rejection episodes (n=10). All transplant recipients are routinely treated with immunosuppressive drug therapy that includes cyclosporin A, azathioprine, and prednisone. Episodes of significant rejection (biopsies demonstrating focal moderate, moderate, or severe rejection) were treated with high-dose intravenous methylprednisolone or oral prednisolone, and in select cases of recurrent or severe rejection, with intravenous OKT3 monoclonal antibody. In studies reported here, however, patients from whom samples were studied were those who did not receive OKT3 monoclonal antibody. The mean age of the transplantation patients was 48 years (range, 15 to 63 years). Donor/recipient MHC class I and II typing results were available for each patient. The samples were selected from 27 patients who had no history of infection and no detectable malignancy. Infection was defined by the presence of (1) positive bacterial or fungal blood cultures or (2) serological or histological evidence of viral infection (eg, cytomegalovirus [CMV]) or protozoan infection (eg, toxoplasma). Percutaneous transvenous EMBs were performed on cardiac transplant recipients within the first week, weekly for 6 to 8 subsequent weeks, and then according to a routine schedule or as clinically indicated.
Peripheral Blood Samples
Heparinized peripheral blood samples
were routinely obtained at
specified time intervals from each patient undergoing cardiac
transplantation at Emory University Hospital. This included samples
procured before transplantation and at scheduled times after
transplantation when EMBs were also obtained or when clinically
indicated. Peripheral blood and lymph node samples from each donor at
the time of transplantation were also obtained. Donor mononuclear cells
were incubated in vitro with supernatant fluid from the B95-8 marmoset
cell line to derive Epstein-Barr virus (EBV)–transformed cell lines.
Donor and recipient cells were typed for MHC class I and II antigens by
the Tissue Typing Laboratory of Emory University Hospital. Aliquots of
PBMCs were cryopreserved at -196°C in media containing 7.5%
dimethyl sulfoxide and 20% pooled normal human AB plasma, and the
samples were tracked with a computerized inventory. All procedures
involving procurement of blood samples and tissues from patients were
approved by the Human Investigations Committee of Emory University
School of Medicine. An informed consent form was signed by each blood
or tissue donor before sample procurement. PBMCs from volunteer healthy
laboratory personnel were used for some of the studies.
Histological Rejection Grades
Aliquots of the biopsies were
fixed in 10% neutral buffered
formalin, processed in a rapid tissue processor in a 4-hour cycle, and
embedded in paraffin as a single block. The biopsies were serially
sectioned, which provides slides at 16 levels. Every other slide was
stained with hematoxylin and eosin, and microscopic evaluation was
performed. Intervening slides were used for special stains if deemed
necessary. The grading of the biopsies was performed according to a
modification of the standardized histological grading system devised by
Billingham et al,33 as described
elsewhere.12 Episodes of significant rejection (biopsies
demonstrating focal moderate, moderate, or severe rejection) were
routinely treated with additional immunosuppression.
Cell Culture
To establish the frequency of
HPRT-
mutants and to define the efficiency of cloning, PBMCs from patients
and healthy control subjects were cultured in vitro, essentially as
described by Albertini et al,30 Allegretta et
al,31 and Gmelig-Meyling et al.32 The medium
for deriving the HPRT- mutant cells consisted
of 47% RPMI 1640, 20% HL-1 medium (Ventrex Laboratories), 20%
lymphokine-activated killer cell supernatant fluid,34 10%
heat-inactivated fetal calf serum (a preselected lot from Hyclone
Laboratories), gentamicin (50 µg/mL), 2 mmol/L
L-glutamine, 25 µg phytohemaglutinin (PHA-16 or -17,
Burroughs Wellcome), and 1x10-5 mol/L 6-TG
(2-amino-6-mercaptopurine, Sigma Chemical Co). For the assay, a
thymidine kinase cell line (TK-) obtained through the
courtesy of Dr R. Albertini (University of Vermont) was used as a
feeder layer. This cell line was routinely screened for mycoplasma and
found to be negative. The TK- cell line was irradiated (8
Gy) with a 137Cs irradiation source (Gamma Cell 40, AEC of
Canada, Ltd), and 1x105 irradiated cells were dispensed in
a volume of 0.1 mL of the above media into individual wells of a
96-well microtiter plate. Twenty-four hours later, PBMCs to be used to
derive HPRT- cells were thawed from
cryopreserved samples and were washed. Then, an aliquot was adjusted to
2x105 cells/mL in the above media. These cells were then
dispensed in a volume of 0.1 mL to each well of a set of five
microtiter plates containing the irradiated TK- cells. A
sixth microtiter plate containing the irradiated TK- cells
alone was included to control for possible reversion and survival of
the 6-TG–resistant cells. In all assays performed, no growth of the
TK- cells cultured alone was observed by 14 days
throughout the studies. The microtiter plates were then incubated at
37°C in a 7%-humidified CO2 incubator and were observed
microscopically for growth of cells at specific time intervals.
Positive fast-growing wells were scored at days 5, 7, and 10 and
distinguished from those that showed visible growth at days 14, 21, or
28. Positively growing cells were expanded and cloned by limiting
dilution assays and used for phenotypic analysis, donor
specificity, and cytokine profile. In the cases in which all wells
containing 6-TG and 2x104 cells showed growth, an aliquot
of the same PBMC sample was diluted, and the assay was repeated using
200, 20, or 2 cells per well to derive more precise evaluation of the
frequency of HPRT- mutant cells.
For each PBMC sample, an aliquot was also subjected to analysis of cloning efficiency (CE). The media that were used for CE determination were identical to those described above except that they did not contain 6-TG. The PBMCs were adjusted for cell concentration so as to yield 0.5, 1, 2, 4, and 8 cells per 0.1 mL of media, and a single 96-well plate was used for each cell concentration. Each microtiter well contained 1x105 irradiated TK- feeder cells, as described above. The microtiter plates were then incubated in the same manner as used to derive the HPRT- mutants, and the frequency of wells showing positive and negative growth at 28 days was recorded. Microtiter wells showing positive growth from each sample at the lowest cell concentration were expanded and then used for phenotypic analysis, donor specificity, and cytokine profile.
In general, an entire series of sequential PBMC samples from a single patient was assayed simultaneously. For purposes of control to provide data on intra-assay variability, an aliquot of a large pool of PBMCs (obtained by leukapheresis of a healthy adult donor) that was cryopreserved was thawed and assayed with each analysis. The frequency of mutant cells and CE that were obtained with this control sample remained constant within 10% SD.
FMC Analysis
Assuming that the limiting dilution analysis of
clonable
cells follows a Poisson distribution, the FMC was established by
dividing the apparent frequency of 6-TG–resistant cells by the cloning
efficiency as follows: FMC=[-ln(fraction of negative wells
in the
presence of 6-TG)/number of lymphocytes per well/CE];
CE=[-ln(fraction of negative wells in the absence of
6-TG)/number of
lymphocytes per well].32 Our standard analysis used
2x104 PBMCs per culture in media containing 6-TG. In
several cases, all five microtiter plates gave positive growth in each
of the 96 wells (ie, 480 positive wells containing 2x104
cells per well or 
50 per 106 cells cultured). In
efforts to derive data on the precise frequency of mutant cells per
106 cells, an aliquot of the same PBMC sample was
further analyzed by use of logarithmic dilutions of 2x104
(ie, 2x103, 2x102, etc).
Phenotypic, Donor-Specific, and Cytokine Profile Analyses
Although these data are not presented in their entirety in
this communication, it is to be noted that each cloned T-cell line was
phenotyped by flow microfluorometry7 and is currently
being analyzed for donor-specific cytotoxic and proliferative responses
by 51Cr-labeled donor EBV-transformed and third-party
non–MHC-related EBV-transformed target cell
lines.7 35
RNA extracted from each cloned T-cell line was assayed for the
presence of message (mRNA) for a battery of cytokines, as
described elsewhere,36 in an effort to designate TH1
versus TH2 phenotype. The presence of mRNA for interferon (IFN)-
but
not interleukin (IL)-4/IL-6 was taken as evidence for TH1, whereas the
presence of mRNA for IL-4/IL-6 but not IFN- was taken as evidence
for a TH2-type clone.37
Statistical Analysis
The objectives of the statistical
analyses described below were
(1) to determine the reproducibility of the method used to count the
frequency of HPRT mutant cells (expressed as
FMC/106 cells); (2) to quantify the time profiles of
FMC/106 cells in serial samples among transplant
recipients; (3) to determine the associations of these profiles with
both the onset of first rejection episodes and, in appropriate
patients, the recurrence of rejection episodes; and (4) to compare
values of FMC/106 cells obtained from samples from
healthy control volunteers, transplantation patients with histology
grades of 0, and transplantation patients with histology grades of 3.
The five patients who did not experience rejection served as the
control group. FMC/106 values obtained within 10
days of treated rejection episodes were excluded from the analysis
because they were likely to be influenced by the immunosuppressive
therapy given.38 Separate models were applied to analyze
the onset of an initial rejection episode and recurrence of rejection
episodes.
To evaluate the reproducibility of the FMC measurements at
low- and
high-frequency levels, repeated-measures ANOVA was applied after
square-root transformations of the frequency data were obtained.
Estimates of measurement error variance (otherwise called
repeatability) and the 95% CIs of reliability coefficients were
determined for the repeated observations.39 40 The
overall
agreement among repeated determinations of FMC/106
values was summarized by the Friedman 
2 test.
For initial rejection episodes, the rising trend of FMC/106 values over serial visits, leading to the first rejection episode, was quantified by fitting a robust regression line to the data collected for each patient.41 The time profiles so determined were then used as data points in subsequent analyses. To compare these robust time profiles in patients with and without rejection, ANCOVA and the Wilcoxon two-sample test were applied, respectively. The association between onset of first rejection episodes and an increased rate of FMC/106 values was examined by logistic regression and Fisher's discriminant analyses.42 Its cross-validated error rate of discrimination (which provides the percent correct rate for identifying onset of rejection episodes between healthy patients and those with rejection) was also determined in the discriminant analysis.
For recurrent episodes, the maintenance of high levels of FMC/106 values after the onset of the initial rejection episode was analyzed. A longitudinal model to Poisson count data was first used to determine the associations between levels of FMC/106 values and recurrent rejection.43 The mean FMC/106 values during recurrent rejection episodes were used to compare with the means determined for control patients by Student's two-independent-sample t test. Similarly, the mean FMC/106 values calculated from the recurrent rejection episodes were contrasted with those determined after the last treated rejection episode by the paired t test. The efficacy of using FMC/106 values to discriminate between healthy patients and those with recurrent rejection episodes was evaluated by the cross-validated error rate determined by the Fisher discriminant analysis method.
Comparisons of FMC/106 values in peripheral blood samples from patients with histological rejection grade 0 and rejection grade 3 versus values from control healthy volunteers were made with an ANOVA followed by a Fisher's PLSD. A value of P<.05 was considered statistically significant.
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Results |
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Validation of the Technique
In efforts to optimize the technical aspects of the assay and to define the reproducibility of the derived data, a first series of studies was conducted in which PBMCs from one healthy individual were obtained at 10 different time periods over a 3-month period, as well as samples from 10 different adult laboratory volunteers obtained on a single day. Each of these samples was assessed for FMC, as described in "Methods." The data showed a mean±SD value of 3.5±1.5 FMC/106 cells (range, 1 to 6 FMC/106 cells) and 3.1±1.45 FMC/106 cells (range, 1 to 6 FMC/106), respectively, for each set of 10 samples. In addition, aliquots of PBMC samples from randomly selected transplant recipients were assayed on 3 different days, and the means±SD of FMC/106 cells were calculated (Table 1
). To assess
the reproducibility of repeated
measurements of FMC/106 values listed in Table 1,
estimates of measurement error variance (otherwise called
repeatability), reliability coefficients, and the Friedman
2 test were performed. Repeated measures in
patients with low frequency values (n=7) and high frequency values
(n=10) gave estimates of measurement error variance of 0.67 and 5.22,
respectively, and reliability coefficients of 0.94 (95% CIs, 0.82,
0.99) and 0.61 (95% CIs, 0.17, 0.90), respectively. The overall
agreement among repeated determinations of FMC/106
values determined by the Friedman 2 test was
P=.55 and P=.145, and the Poisson variance
test
ranged from 0.78 to 0.99 and 0.1 to 0.83, respectively.
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In efforts to ensure that the frequency analysis was based on statistically valid culture conditions, cells from patients and control subjects were cultured by limiting dilution analysis in the presence and absence of 6-TG, and data were derived on the fraction of microtiter culture wells that showed growth at each cell concentration. Data showed an excellent log-linear relation between cell input and fraction of negative wells (data not shown), similar to the results published elsewhere.32 The cloning efficiencies ranged between 5% and 92% for healthy control subjects and between 4% and 74% for the patients.
Correlation of Rejection Grade With FMC
In efforts to
evaluate this technique, a second series of studies
was conducted in which aliquots of cryopreserved PBMC samples that
corresponded with a rejection grade of 3 were compared for the
frequencies of HPRT- mutants per
106 cells with the frequencies derived from a
sequential series of PBMC samples from 5 patients with no history of
rejection (the two extremes). In addition, PBMC samples from healthy
adult volunteers were included as control subjects for the evaluation
of the data. As shown in Fig 1, PBMC samples from
healthy control subjects (n=10) and patients with no history of
rejection (grade 0, n=32) gave values 
6 FMC/106
input cells. In contrast, 19 of the 22 PBMC samples with rejection
grade of 3 gave values >6 FMC/106 cells, 14 of
which gave values of >20 FMC/106 cells. Of these
14, 11 gave values of >50/106 cells (range, 146 to
46 982 FMC/106). Statistical analysis of the
data revealed that values of FMC/106 input cells on
PBMC samples from healthy control subjects (n=10) versus those from
patients with no histological evidence of rejection (n=32) gave a value
of P=.9474 (not significant). However, the value for data
comparing samples from patients having grade 3 rejection with samples
from control subjects and from patients with no histological evidence
of rejection (n=32) was P<.0001.
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Sequential Studies of PBMC Samples
The third series of
studies used previously cryopreserved
sequential PBMC samples from patients after transplantation. Samples
were chosen corresponding to the recorded history of histological
rejection grades that were thought to be informative. This study thus
comprised a series of at least 6 consecutive specimens from 5 patients
(patients 23 through 27, n=32) who had a recorded rejection grade of 0
after transplantation; a series of at least 10 consecutive specimens
from 6 patients (total, 64 specimens) who experienced a single
rejection episode early after transplantation (<1 year after
transplantation); a series of at least 7 consecutive specimens from 6
patients (total, 56 specimens) who experienced a single rejection
episode late after transplantation (>1 year after transplantation);
and a series of at least 10 consecutive specimens from 10 patients
(total, 141 specimens) who experienced multiple rejection episodes.
The
data obtained on the 32 samples from patients 23 through 27 with no
history of rejection are shown in Fig 1
. The profiles of data
from
sequential PBMC samples from the 6 patients (patients 1 through 6) who
experienced a single rejection episode early after transplantation and
from 6 patients who experienced a single rejection episode late after
transplantation (patients 7 through 12) are illustrated in Fig
2A and 2B, respectively. Similarly,
profiles of data from sequential PBMC samples of 10 patients (patients
13 through 22) who experienced multiple rejection episodes are
illustrated in Fig 3A and 3B. Please note that
since the
PBMC samples of 4 of these 10 patients (patients 13 through 16) with
multiple rejection episodes gave a wide range of values, this could be
depicted only with a log scale for the y axis (see Fig
3A),
whereas profiles of the data of the sequential PBMC samples from the
other 6 patients (patients 17 through 22) who experienced multiple
rejection episodes are shown in Fig 3B with a linear scale for
the
y axis.
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Single Rejection Episodes
As illustrated in Fig
2A, sequential PBMC samples from each of 6
patients who experienced a single rejection episode relatively early
after transplantation, as defined by histological grading of their
corresponding biopsies, showed rising numbers of mutant cells
(FMC/106) before the rejection episode. It should be
noted that although there is considerable variation in the absolute
numbers of FMC/106 with samples from individual
patients (eg, samples from patients 1 and 2 show relatively lower
FMC/106 values than do patients 3 through 6), in
each case, an increase in the FMC/106 value predicts
the rejection episode. In addition, in 5 of these 6 patients, the PBMC
sample immediately following the rejection episode showed a marked
decrease in the FMC/106 value. In patient 3, this
decrease was noted in the second sequential sample after the rejection
episode. These decreases in FMC/106 values
presumably are secondary to successful response to immunosuppressive
therapy.
Analysis of the profiles of FMC/106 values obtained from sequential PBMC samples of patients who experienced a single rejection episode relatively late after transplantation (patients 7 through 12) showed some differences from profiles of samples from patients early after transplantation (patients 1 through 6). While the FMC/106 values in samples preceding the rejection episode showed marked increases predictive of the rejection episode, the absolute number of FMC/106 was considerably higher in samples from patients 7 through 12 compared with patients 1 through 6. (Note that although the value of the sample on day 1056 of patient 10 appears low, the precise value was 112 FMC/106 compared with 12 FMC/106 in the preceding day 952 sample.) Consistent with the findings of data from patients 1 through 6, samples from patients 7 through 12 immediately after the rejection episode showed a marked decrease in 5 of the 6 patients. The other important difference noted was that, in some cases (patients 8, 9, and 10), the increase in the FMC/106 value was noted up to 100 days or more before histologically diagnosed rejection. It is our working hypothesis that this prolonged high frequency of putatively activated cells represents chronic immune activation that may be an ongoing process whose effector function may be regulated locally in situ by immune mechanisms mediated by cellular infiltrates present in the donor allograft. A possible source of activated T cells detected over prolonged periods is the subendothelial infiltrates (endothelialitis). Such infiltrates, reported by many laboratories, are often detected in EMB specimens, even in the absence of myocardial rejection. In each of the cases studied, the FMC/106 cells eventually returned to values of <6 FMC/106 cells after histologically diagnosed rejection, most likely secondary to successful immunosuppressive therapy.
Recurrent Rejection Episodes
The next series of studies
centered on analysis of data on
sequential PBMC samples from patients who experienced multiple
rejection episodes. The profiles of the data from sequential samples
from the 10 patients are illustrated in Fig 3A and
3B. (Note the use of
logarithmic scale for the y axis in Fig 3A and the
linear
scale for Fig 3B, which was used because of the variability in
the data
obtained.) Of great interest was the finding that in addition to an
increase in the FMC/106 cells before histological
rejection in each case (although some variation was noted), it appeared
that elevated values were maintained after the first rejection episode.
These data are interpreted as an indication that the patients were most
likely refractory to immunosuppressive therapy. Note the data obtained
on sequential samples from patient 13. Samples after the second
rejection episode, especially day 174 and beyond, continued to give
high FMC/106 values. The patient eventually died on
day 276. In the other 9 patients, the values of
FMC/106 eventually returned to <10
FMC/106, providing suggestive evidence for
the final resolution of the rejection response.
Statistical Analysis of FMC/106 Values Over a
Period of Time
To quantitatively express the profiles of
FMC/106 values, we initially included all patients
with rejection (n=22) and examined the trends (determined by robust
linear regression slopes) in FMC/106 values in
samples before the onset of initial rejection compared with
FMC/106 values from serial samples in patients
without rejection (11.53±1.33 versus 0.13±0.09,
P=.002 by
ANCOVA and by Wilcoxon two-sample test). We then determined that
values of FMC/106 decrease after successful
immunosuppression for rejection (slope, -5.38±1.12,
P=.0001 by ANCOVA and by Wilcoxon two-sample test compared
with slopes before initial rejection). Last, a highly significant
association between the onset of first rejection and an increased rate
of FMC/106 values (determined by the logistic
regression method) was noted (P=.0001), after adjustment for
age and sex. Based on the cross-validated error rate determined by
Fisher's discriminant analysis, the ability of a rising trend in
FMC/106 values to correctly identify the onset of
rejection was 81.8% and to correctly identify no rejection, 100%.
For the subgroup of patients with recurrent rejection episodes (n=10), trends of FMC/106 values during recurrent episodes were significantly higher than with patients without rejection episodes (37.57±4.57 versus 2.75±0.51, P=.0001 by Student's two-independent-sample t test and by Poisson regression). Thus, a rise and/or consistent elevation of values of FMC/106 cells appears to be associated with incomplete immunosuppression and subsequent episodes of rejection.
Trends of FMC/106 values during recurrent rejection episodes were also significantly higher than those that followed the last treated rejection episode (37.57±4.57 versus 10.24±1.35, P=.004, paired t test), suggesting that FMC/106 values drop after successful immunosuppressive therapy for rejection. In addition, a highly significant association between the recurrent rejection episodes and the maintenance of increased rates of FMC/106 values (determined by the logistic regression method) was noted (P=.0003) after adjustment for age and sex. On the basis of the cross-validated error rate determined by Fisher's discriminant analysis, the ability of a persistently elevated trend in FMC/106 values to correctly identify recurrent rejection was 90% and to correctly identify no rejection, 100%.
Characterization of the Mutant Cells
An extensive database is
continuing to be acquired on the
phenotype of each of the mutant cultures (n>2000), their donor helper
T lymphocyte (HTL) and CTL specificity, and cytokine profile
(determined by reverse transcriptase–polymerase chain reaction). These
data are being prepared for a separate communication. In brief,
however, the data indicate that the HPRT-
mutants isolated from the PBMCs of patients undergoing a rejection
episode early after transplantation (<1 year) have a dominant
CD8+ phenotype, whereas HPRT-
mutants from patients in the late posttransplantation period are
predominantly CD4+. In addition, there is a decrease in the
frequency of donor-specific HPRT- CTL in the
PBMCs of patients from both groups, which is coincident with rejection
(see Table 2). The cytokine profile of the
donor-specific HPRT- HTL in both groups was
consistent with a TH2 phenotype, but the donor-specific
HPRT- CTL gave mixed TH1/TH2 profiles (see
Table 3).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Although cardiac transplantation is being performed routinely in many clinical centers around the world, there is an
20% mortality
rate at 1 year after transplantation (primarily due to
infection/rejection) and 40% by 5 years after transplantation
(primarily secondary to other complications such as accelerated graft
atherosclerosis6 44 ). According to the 1994
International
Society of Heart and Lung Transplantation, 26 704 heart
transplantations have been performed at 251 centers. In addition, 1567
heart-lung transplantations have been performed at 93 centers.
Currently, 10 330 transplant recipients are registered at 173 centers
worldwide,45 and the number of recipients is slowly but
gradually increasing because of improved patient survival rates. These
patients are currently monitored for rejection for their lifetime by
repeated cardiac biopsies that are histologically graded by criteria as
modified by Billingham et al.33 As described above, there
are several limitations to the use of EMB specimens, including the
invasiveness of the procedure and its associated risks, sampling error
due to nonuniform distribution of myocardial lesions, and the
subjectivity inherent to biopsy interpretation. In addition, full-blown
rejection episodes cannot be predicted in advance, since biopsies
showing moderate or severe rejection are often preceded by
"normal" biopsies. For these reasons, a number of investigations
have been directed toward developing noninvasive procedures for the
diagnosis of rejection. Studies have analyzed electrophysiology,
echocardiography, biochemical markers, radioisotope techniques,
magnetic resonance imaging, and a battery of immunologic tests
(reviewed in References 5 and 6). However, no noninvasive technique
thus far has demonstrated sufficient sensitivity or specificity to
replace the need for EMB in the diagnosis of rejection.
The noninvasive immunologic parameters that have so far been examined
include the monitoring of levels of IL-2R,46 expression of
transferrin receptors,47 quantification of tumor necrosis
factor (TNF)-
levels,48 and studies aimed at
quantifying the frequency of donor-specific HTLs and CTLs by limiting
dilution
analysis,28 49 50 51 52 53
to name a few. The daily
fluctuations of IL-2R and transferrin receptor levels and their lack of
correlation with EMB-based rejection episodes and lack of specificity
in distinguishing infection from rejection have led to healthy
skepticism regarding the feasibility of replacing EMB with these
indicators. Clearly, such studies may be important adjuncts for making
decisions regarding the institution of immunosuppressive therapy. High
quantitative levels of serum TNF- correlate with severe episodes of
humoral but not cellular rejection.48 In regard to
quantitative analysis of donor-specific HTL and CTL frequencies in
the PBMCs of transplant recipients, in general, it appears that the
majority of studies report a high correlation between prolonged graft
survival and a decrease in donor-specific reactivity (donor-specific
hyporesponsiveness).49 51 52 54 55 56
It has been reasoned that such decreased reactivity could be due to selective entrapment of donor MHC-specific clones within the graft, induction of "anergy" within the donor-specific T-cell clones in circulation, or regulatory mechanisms that remain ill-defined. Analysis of our data on the unmanipulated recipient PBMCs for donor-specific reactivity is basically in agreement with these findings. There did not appear to be any correlation between donor-specific HTL or CTL frequency and histological grades of rejection, as noted elsewhere.53 There are, however, reports of an association between increased donor-specific HTLs and allograft rejection.50 The reasons for these differences are not readily apparent. The concept of entrapment of donor-specific T-cell clones in the allograft has prompted several laboratories, including ours, to study such graft-infiltrating cells for donor-specific frequencies and specificity. Data from such studies certainly support the view that there is selective enrichment of donor-specific T-cell clones in grafted tissues.22 23 24 25 26 27 28 29 Detailed analysis of cells cultured from allograft biopsies for donor-specific proliferative responses called HTLs, donor-specific sensitized (committed) CTLs (c-CTLs), and those with potential to be cytolytic for donor MHC (p-CTLs) has been carried out by use of the paradigm by Orosz et al57 for distinguishing such cells. These studies, however, require a biopsy specimen.
Potential Importance of Findings
The studies reported in this
communication consisted of using an
assay that has been studied in detail by a number of laboratories.
These studies have focused on optimizing culture conditions;
determining cell concentrations to be used; analyzing a number of
variables such as cloning efficiency, the effect of age and sex of the
blood donor, and the effect of cell cycle; and assessing the influence
of cytotoxic drugs, drugs used for chemotherapy of cancer patients, and
radiation
therapy.30 58 59 60 61
Our laboratory used this
information and optimized the culture conditions and assay, and we
demonstrate here that (1) the assay is reproducible, (2) a rising trend
in the values of FMC/106 in sequential samples can
discriminate between patients experiencing the onset of an initial
episode of rejection and patients without rejection, and (3) a
sustained increase in the frequency of HPRT-
mutant cells/106 is found in patients who experience
recurrent rejection. Moreover, sustained increases in the frequency of
HPRT- cells support the notion that these
patients have substantial in vivo immune activation in progress that
does not respond to standard posttransplantation immunosuppressive
therapy protocols. To adequately assess the scope of clinical
applicability of the assay described in this communication, several
questions need to be addressed. First, can we distinguish
infection-related immune activation from true allograft rejection?
Second, what is the influence of the immunosuppressive drugs that are
administered to patients after transplantation? Third, what do these in
vivo activated HPRT- mutant cells
represent? Fourth, what is the basis for their increased
frequency in some patients for prolonged periods of time before
histologically diagnosed rejection and in other patients after a
rejection episode? Fifth, is this assay applicable to only cardiac
organ allografts? Sixth, why does the frequency of
HPRT- cells finally decrease? Finally, does the
turnaround time for obtaining results from the assay limit its clinical
utility for the assessment of transplantation patients prospectively?
It is our working hypothesis that the response of the host to
allografts is highly complex, dynamic, and variable, depending on a
number of factors, including the degree and type of MHC incompatibility
between donor and recipient and individual variability in the
immunologic pathways involved in allograft rejection (direct versus
indirect), the role of regulatory cells, and the susceptibility of the
effector and regulatory cells to immune suppression with the drugs
currently being used.
In regard to infection-related immune activation
and allograft
rejection, our laboratory has studied sequential PBMC samples from two
patients with CMV (patients 28 and 29) and one patient with toxoplasma
infection (patient 30), documented by rising IgG antibody titers
against CMV and histological diagnosis from EMB specimens in one CMV
patient and the patient with toxoplasma infection. These patients and
their samples were chosen because we had cryopreserved PBMCs that
included three sequential samples before and after diagnosis of
infection and sequential samples from these patients at a later time
after transplantation (>6 months) when each experienced a histological
grade 3 rejection episode. Thus, we could compare the
FMC/106 cells from the same patient during infection
and rejection. The data derived from these studies confirmed, first,
our previous findings that an increase in the frequencies of
FMC/106 cells preceded the histological diagnosis of
rejection in the EMB specimen in each of the three patients studied
(see Fig 4). Second, the frequencies of
HPRT- mutant cells/106 PBMCs
were significantly higher during the rejection period in each case
compared with values of FMC/106 cells before,
during, and after diagnosis of infection in each case (see Fig
4).
Third, phenotypic analysis of the mutant cultures showed the
predominance (>75%) of CD8+ T cells during the infection
episode compared with the predominance (>90%) of CD4+ T
cells during the rejection episode (data not shown). Last, cytokine
analysis of the mutant cultures showed a predominant IFN-
profile during infection (TH1 type) compared with the predominance of
IL-4/IL-6 and the absence of IFN- mRNA profile during rejection (TH2
type) (data not shown). Although these data are preliminary, they
provide the basis for further exploration. Certainly, patients need to
be studied to establish whether the assay to measure the frequency of
HPRT- mutants can discriminate between
infection-related activation and immune rejection. Our preliminary data
suggest that, independent of the frequency of mutant cells, it might be
possible to distinguish infection-related activation from rejection by
using concordant sets of other data, such as cell surface phenotype and
cytokine profile, in combination with serological data and studies of
the biopsy for the presence of infection.
|
Previous studies have focused on the effect of various forms of drugs and agents in inducing mutations such as the HPRT- gene in the PBMCs of patients. These drugs and agents include steroids for the therapy of lupus,32 antineoplastic agents, irradiation,59 and addictive drugs60 in inducing mutations in the PBMCs of patients. In addition, the possibility that the patients are carriers of a partial defect in the HPRT- gene was entertained. In regard to the effect of drugs, it is clearly possible that the immunosuppressive drugs administered may influence the frequency of HPRT- mutant cells, especially if they are effective on dividing cells. Clearly, values obtained on samples from patients in the late posttransplantation period are in most cases much higher than on samples from patients in the early posttransplantation period. Nonetheless, the possibility of an additive drug effect cannot be ruled out. However, since variable frequencies are obtained on multiple samples from the same patient, these frequencies cannot account for a defect in the HPRT- gene, since the frequencies of HPRT- mutant cells, in this case, would always be high. In addition, since all patients who were studied, including those who experienced no rejection, receive standardized immunosuppressive drug regimens and since the frequencies of HPRT- mutants in sequential samples from the same patient change, it is unlikely that the increase in mutant cell frequencies is the manifestation of drug effects.
In regard to the nature and function of the HPRT- mutants, as noted above, our laboratory has prepared cloned T-cell lines from both the HPRT- mutant cultures and the cloning efficiency cultures that were performed on each PBMC sample. The cloned T-cell lines have been phenotyped for both cell surface markers and the pattern of cytokines they synthesize. In addition, the frequency of cloned T-cell lines that demonstrate donor-specific proliferative and cytotoxic response is being characterized. The results of these studies are being prepared for a separate communication.
As seen in Fig
3A and 3B, PBMC samples from some patients show
prolonged increased levels in the frequency of
HPRT- mutants either before or after a
rejection episode. While continued allogenic stimulation that is
insensitive to the immunosuppressive drugs being taken by the patient
may account for such continued increased frequencies of
HPRT- mutants in those who have multiple
rejection episodes, it is difficult to determine the reasons for such
sustained increases in patients who experience a single rejection
episode. It is possible that such increased in vivo activation
represents host regulatory responses involved in graft
"adaptation," which uses pathways of T-cell activation that are
not sensitive to immunosuppressive drugs. The composite functional
analysis of such cells, which is in progress, may shed light on
this issue. It should be noted that the occurrence of sustained
increased levels of HPRT- mutant cells in the
PBMCs has also been noted in patients whose biopsies showed no evidence
of rejection but who died secondary to accelerated graft
atherosclerosis (AGA) after cardiac transplantation (personal
observation). Thus, sequential PBMC samples from these patients up to
the time of their death showed >50 FMC/106 cells.
Data from one such patient (patient 13) are shown in Fig 3A.
The
specificity and function of cloned T-cell lines established from such
in vivo activated HPRT- mutant cells from the
PBMCs of these patients who died from AGA are currently under study to
establish the role of the mutant cells in the induction and/or
perpetuation of immune-mediated AGA.
On the other hand, the mechanisms
by which the frequencies of
HPRT- mutants eventually decrease are also not
clear at present. It is possible that this decrease may be related
to the pathways used by T cells and the ratio of various T-cell subsets
that are mediating rejection and their relative susceptibility to the
immunosuppressive drug therapy being administered. In this regard, if
immunosuppressive drugs inhibit the maturation of donor-specific p-CTL
to c-CTL and are not effective in inhibiting the effector function of
c-CTL, the rejection response will continue until the frequency of
c-CTL is exhausted. Studies of the precise pathways of intracellular
drug-induced inhibition of T-cell subsets may promote our understanding
of this important issue. In regard to the ability to obtain meaningful
data from this assay in a timely fashion for prospective use, it is
important to note that significant increases in the number of
microtiter wells containing HPRT- mutant cells
could be visualized as early as day 5 (see Table 2) in select
samples
of PBMCs that contained high frequencies (>75%) of CD8+ T
cells during the infection episode compared with the predominance
(>90%) of CD4+ FMC/106 cells of
HPRT- mutant cells (see Table 2).
Thus, for
most scheduled patient evaluations, such an assay could be used
prospectively. In addition, our laboratory is using more sensitive
assays for detection of cytokines in the supernatant fluid, for
example, which may facilitate obtaining data within a more favorable
time period to maximize the clinical utility of the data. As far as the
application of this assay for defining frequencies of
HPRT- mutants in the PBMCs of patients
undergoing organ transplants other than heart is concerned, our
laboratory has not studied this issue.
Conclusions
In summary, these data provide the basis for
examining the
applicability of this assay to cardiac transplant recipients and, at a
minimum, may be a valuable adjunct to data derived from studies of EMB
specimens. Assessment of the clinical utility of this assay in its
present form, in a prospective study of a larger group of patients,
is in progress.
|
|
Acknowledgments |
|---|
The authors are deeply grateful to the professional and technical staff of the Emory University Hospital cardiac transplantation team, especially Corby D'Amico and Dawn Rikard. In addition, the authors are grateful for the zeal and effort of Carey Clutts, Dirk Hunt, Mark Meadows, and Dan Clark in performing the massive number of assays required to derive the data presented above. This work was supported by grant 1R01-HL-47272-06 from the National Institutes of Health.
|
Footnotes |
|---|
Reprint requests to Dr A.A. Ansari, Winship Cancer Center, 1327 Clifton Rd NE, Atlanta, GA 30322.
Received January 10, 1995; revision received February 2, 1995; accepted February 10, 1995.
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References |
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