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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3990-3995
TRANSPLANTATION
Longitudinal monitoring of immune reconstitution by CDR3 size
spectratyping after T-cell-depleted allogeneic bone marrow transplant
and the effect of donor lymphocyte infusions on T-cell repertoire
Stephanie Verfuerth,
Karl Peggs,
Paulomi Vyas,
Lorna Barnett,
Richard J. O'Reilly, and
Stephen Mackinnon
From the Department of Haematology, University College London,
London, United Kingdom; and Bone Marrow Transplant Service, MSKCC,
New York, NY.
 |
Abstract |
Delayed immune reconstitution after allogeneic bone marrow
transplantation (BMT) with associated infection is a major cause of
morbidity and mortality. We used third complementarity region (CDR3)
size spectratyping as a tool for monitoring T-cell repertoire reconstitution in 19 patients over a median time of 40 months after
T-cell-depleted allogeneic BMT for chronic myeloid leukemia (CML).
Furthermore, the effect of donor lymphocyte infusions (DLI) for the
treatment of relapse in 18 of the 19 patients was analyzed. All BMT
recipients had irregular spectratypes in the first 3- to -6 months
after transplant. These evolved to more normal patterns by 12 months
after transplant and continued to improve thereafter. In approximately
a third of the patients, it took 2 to 3 years for all spectratypes to
normalize, whereas in the other two thirds, some abnormal spectratypes
persisted even after several years. In 9 patients, there was no
immediate change in the CDR3 size profiles after DLI. In 3 patients,
spectratypes improved slightly after DLI, whereas in 6 patients,
spectratypes became more restricted and irregular. Overall, T-cell
spectratypes in BMT patients were characterized by instability over
time and in patients with graft-versus-host disease (GVHD), this was
even more exaggerated. Several factors, such as pre-BMT conditioning,
T-cell depletion of the donor marrow, loss of thymic function in
adults, exposure to infectious agents, GVHD, and immunosuppressive
treatment, are likely contributors to the delay in T-cell-repertoire reconstitution.
(Blood. 2000;95:3990-3995)
© 2000 by The American Society of Hematology.
| |
Introduction |
Although allogeneic bone marrow transplantation (BMT)
from HLA-matched donors can provide a curative therapy for chronic
myeloid leukemia (CML)1 and other hematologic disorders,
the long period of immune compromise experienced by BMT recipients
after transplant is a major cause of morbidity and mortality in this
patient group. T-cell immunity is affected most, because the T-cell
pool is largely eliminated by the pretransplant conditioning, and the
capacity for thymic output is very limited in adults.2,3
Thus, T-cell immunodeficiencies render BMT recipients susceptible to a
number of life-threatening opportunistic infections.4,5
T-cell-repertoire reconstitution can be further delayed by the
development of graft-versus-host disease (GVHD).6
Removal of T cells from the donor bone marrow before transplant reduces
the risk of GVHD by limiting the number of allo-reactive T cells. An
increased risk of relapse is, however, associated with the use of
T-cell-depleted bone marrow compared with unmanipulated bone marrow,
because T cells involved in graft-versus-leukemia (GVL) responses are
also removed.7,8
Donor lymphocyte infusion (DLI) is an effective treatment for patients
with relapsed CML. It induces GVL responses and results in complete
remission in the majority of patients.9 However, DLI
can also cause GVHD. Whether GVL occurs without GVHD depends to a
large extent on the dose of donor lymphocytes infused.10
The technique of third complementarity region (CDR3) size analysis
developed by Cochet et al11 and later called
"spectratyping" by Gorski et al12 is a powerful tool
to analyze T-cell receptor (TCR) diversity in health and disease.
Reverse transcriptase-polymerase chain reaction (RT-PCR) products
spanning the constant (C) /variable (V) region junctions of the CDR3
beta (B) chain in the TCR are separated according to size, giving rise
to a spectrum of different size classes for each of the 22 BV gene
families expressing functional rearrangements, thus providing a finer
level of resolution than just analyzing BV gene family expression.
CDR3 beta chain size heterogeneity arises during developmental DNA
rearrangement when a BC segment is joined with 1 of 22 BV segments via
a diversity (D) and a joining (J) segment to give rise to a complete BV
gene. During this process, random numbers of nucleotides are inserted
and deleted enzymatically at the junctions between the gene
segments.13 Successful rearrangements differ in size by
multiples of 3 nucleotides. Any other rearrangements would be out of
frame and are rarely detected in peripheral blood T
cells.12 As the ratio of transcripts per cell is fairly
constant,14 the amount of PCR product in each size class
gives an indication of the number of clonotypes and thus T-cell
repertoire diversity. Predominance of only a few size classes in a
spectratype would indicate oligoclonality. Gaps in a spectratype
indicate a lack of T cells expressing a certain CDR3 size class.
However, sequencing would be required to determine the number of clones
that make up each size class.
Several authors have successfully used CDR3 size spectratyping to study
aspects of T-cell-repertoire reconstitution after BMT. Gorski et
al12 related T-cell-repertoire complexity in BMT
recipients to their state of immune function, Dietrich et al15 examined T-cell spectratypes in GVHD skin lesions, and Akatsuka et al16 and Roux et al17,18 looked at
differences in T-cell-repertoire reconstitution in recipients of
T-cell-depleted and -undepleted bone marrow. Finally, Claret et
al19 characterized T-cell repertoires in BMT recipients
with GVL responses after DLI. Limitations of these studies were
the small number of patients included and short intervals of time after
transplant over which patients were followed.
Here, the T-cell repertoires of 19 allogeneic T-cell-depleted BMT
recipients were analyzed by CDR3 size spectratyping longitudinally for
a median time of 40 months after BMT for CML. Furthermore, 18 of these
patients underwent DLIs at some stage after BMT for treatment of
relapse of their diseases. The effect of DLI on the T-cell repertoire
was analyzed.
| |
Patients, materials, and methods |
Patients
Nineteen patients with CML in the first chronic phase were
studied after allogeneic BMT. Donor-recipient pairs were matched at the
human leukocyte antigen A, B, and DR loci (Table
1). The patients were conditioned
with fractionated total body irradiation 12 × 125 cGy + cyclophosphamide 120 mg/kg + thiotepa 10 mg/kg. All patients received a
T-cell-depleted marrow graft. In vitro T-cell depletion was performed
using soybean agglutinin (SBA) to produce an SBA-marrow
fraction. This was further T-cell depleted by rosetting
with sheep erythrocytes to give a SBA-E-marrow.20 Eighteen of the patients received DLIs for the treatment of
relapse as previously described.10
RNA extraction
Peripheral blood mononuclear cells (PBMCs) were obtained from
heparinized blood by density gradient centrifugation through Ficoll-Paque (Pharmacia Biotech, St Albans, UK). RNA was
extracted from the cells using Ultraspec RNA (BiotecX Laboratories,
Houston, TX), according to the manufacturer's protocol.
Briefly, 5 to 10 million cells were homogenized in Ultraspec, followed
by the addition of chloroform to separate the organic and aqueous
phases. RNA was precipitated from the aqueous phase with isopropanol,
followed by a wash with 70% ethanol. The resulting RNA pellet was
resuspended in sterile water, and the yield of RNA and purity
were determined by spectrophotometry.
Reverse transcriptase reaction
Complementary DNA (cDNA) was generated from 1 µg of RNA in a 20 µL reaction using random hexanucleotide primers for reverse transcription with reverse transcriptase (Superscript,
Gibco, Paisley, UK).
Polymerase chain reaction primers
Each of 22 functionally rearranged BV gene subfamilies was amplified
across the C/V junctions using the 24 BV subfamily-specific primers
described previously by Maslanka et al,21 and a fluorescent dye (FAM, Perkin Elmer)-conjugated BC region-specific
primer.12 Some of the BV primers amplify short, and others
longer PCR products. "Short" and "long" BV primers were
combined in multiplex PCRs to amplify 2 BV families in 1 reaction as
follows: BV 5.1 + 1; BV2 + 12; BV8 + 3; BV4 + 5.4; BV13 + 7;
BV9 + 14; BV11 + 20; BV17 + 15; BV16 + 21; BV18 + 23; and
BV24 + 22. BV6.1 and BV6.2 were used unpaired.
Polymerase chain reaction
The total PCR volume was 20 µL containing Genamp PCR buffer
(Perkin Elmer), 2 mmol/L MgCl2, 0.2 mmol/L each dNTP, 1 mmol/L of each primer, and 1 µL cDNA (equivalent to approximately
25 000 cells). For the hot start, 0.5 units of Amplitaq DNA polymerase were added last, after a 5-minute denaturation step at 95°C.
Optimal cycling conditions were 95°C for 30 seconds, 58°C for
30 seconds, and 72°C for 45 seconds, for 30 cycles, followed by a
final extension at 72°C for 5 minutes.
T-cell spectratyping
One microliter of PCR product was denatured in 12 µL formamide and
electrophoresed through Performance Optimized Polymer 4 (Perkin Elmer,
Cambridge, UK) on an ABI 110 automated sequencer (Perkin Elmer) in the
presence of Tamra 500 size standard (Perkin Elmer). Genescan software
2.1 (Perkin Elmer) was used to analyze the data. For the purpose of
analyzing the T-cell repertoire of BMT recipients, a normal spectratype
was defined as comprising at least 6 different size classes, at
intervals of 3 nucleotides without any gaps.
T-cell dilution experiments
PBMCs from a healthy young donor were separated by density gradient
centrifugation through Ficoll-Paque. The T2 lymphoblastic hybridoma
cell line, which expresses TCR BV 9, and the Jurkat T-cell line, which
expresses TCR BV8, were used to spike these polyclonal PBMCs in
separate experiments. Serial dilutions of each cell line in PBMCs were
made from 1:100 down to 1:100 000. A total of
1 × 107 cells of each dilution was used for RNA extraction.
| |
Results |
Definition of abnormal CDR3 size profiles and sensitivity of CDR3
spectratyping
TCR CDR3 size profiles in BMT recipients were compared with those in
healthy adults. Although the amount of product in each size class in a
normal spectratype is typically distributed in a Gaussian fashion with
6 to 10 different size classes at 3 nucleotide intervals, spectratype
shapes frequently deviate markedly from this normal distribution
pattern by having 1 or 2 predominant size classes.22 Hence,
in this study, normal spectratypes were defined as consisting of at
least 6 peaks spaced 3 nucleotides apart without any gaps.
T-cell numbers are often low in BMT recipients, especially shortly
after transplant. In healthy individuals, l0 000 cells produce normal
spectratypes (results not shown). In our PCRs, we used an amount of
cDNA equivalent to approximately 25 000 PBMCs, which is in excess of
the limiting cell number. The deviations from a normal Gaussian
distribution in healthy individuals are accounted for by clonal
expansions of T cells in the CD8+ subset.23
Hence, a spectratype analysis of the CD8+ subset could have
been more informative. However, because of the low cell numbers
available at certain time points and additional losses that would have
occurred during processing, it was decided not to separate samples into
subsets for analysis.
The sensitivity of the CDR3 spectratyping technique was determined by
spiking experiments using the T2 and Jurkat cell lines. Analysis of the
spectratype profile of spiked normal PBMCs showed that the clones can
be detected at a dilution of 1:1000, but not 1:5000 or above (Figure
1). This confirms published
results.24,25
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| Fig 1.
Determination of spectratype sensitivity to the addition
of cells bearing clonally rearranged T-cell-receptor genes.
Serial dilutions of both Jurkat and T2 cell lines were made using PBMCs
from the same healthy donor. In both cases, the threshold for detection
lies between 1:1000 and 1:5000.
|
|
CDR3 size profile evolution in allogeneic T-cell-depleted bone
marrow transplant recipients
T-cell repertoires of 19 BMT patients were monitored by TCR
spectratyping over a median time of 40 months after transplantation. The aim was to determine how long it takes after BMT for the T-cell repertoire to reach normal polyclonal complexity. At least 6 BV subfamilies were amplified and analyzed for each BMT recipient. The
primer sets chosen differed among the various patients, however, subfamilies overrepresented and underrepresented within the normal repertoire were included for each patient. In most patients, all BV
subfamilies comprised only very few size classes during the first 3 months after BMT (example in Figure 2A,
first time points after BMT). After 6 months, T-cell repertoires
gradually diversified. By 12 months, profiles of some BV subfamilies
contained a full set of size classes in nearly all patients. However,
transient changes in the amount of product in each CDR3 size class were common (eg, Figure 2, early time points). Furthermore, in all patients,
some very abnormal spectratypes persisted by 12 months after BMT.
Between 2 and 3 years after transplant, all patients demonstrated at
least some normalization of CDR3 size profiles. However, there were
marked differences in the progress of CDR3 size profile normalization
between different patients as well as between the different BV
subfamilies in the same patient. This is illustrated in Figure 2A,
showing examples in 2 BMT recipients of normalizing (BV13 and 2) and
abnormal (BV9 and 24) series of CDR3 size spectratypes.
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| Fig 2.
Examples of CDR3 size pattern evolution after BMT.
Size profiles of 2 different BV subfamilies at 6 time points are shown
for each patient. (A) Examples of interpatient and intrapatient
variability in patients 15 and 17, who underwent DLIs +13 months and
+14 months after BMT, respectively. (B) Examples of spectratype change
after DLI. Improving spectratypes in patient 3 (DLI +22 months) and
more abnormal spectratypes after DLI in patient 11 (DLI +9 months).
|
|
In 6 patients, all spectratypes analyzed became similar to those of
healthy adults. Figure 3 illustrates the
normalization of the size profiles with 21 BV-specific primers between
13 and 37 months after BMT in patient 1. In the remaining 13 patients, some BV spectratypes normalized, whereas others were characterized by
oligoclonal patterns that persisted until the end of the study, which
meant up to 5 years after BMT in some patients.
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| Fig 3.
CDR3 size profiles with 21 subfamily-specific primers at
2 time points after BMT in PBMCs from patient 1.
The patient underwent DLI +23 months after BMT, with subsequent
normalization of spectratypes by +37 months after BMT.
|
|
Eighteen of the 19 patients underwent DLIs to treat relapses. Sixteen
of these patients achieved remission after DLIs (Table 2). In 9 patients, there was no detectable
change in T-cell spectratypes between the pre-DLI and early post-DLI.
In only 3 patients, the spectratypes became slightly more normal
(example shown in Figure 2B, patient 3), whereas in 6 patients, the
CDR3 size patterns appeared more abnormal after DLI (example shown in
Figure 2B, patient 11). As described above, in the longer term, T-cell
repertoires did improve, but whether this was the result of a delayed
indirect effect of DLI or independent of DLI remains unclear. The
timing of DLI after BMT did not seem to influence its effect on the
T-cell repertoire. Whether an association exists between spectratype normalization and chimeric status was not clear. In the 2 patients who
retained mixed chimerisms after DLI (Table 2), spectratypes became more
abnormal in 1 (Figure 2B, patient 11), whereas no marked change in
spectratypes after DLI occurred in the other.
In 4 of the 18 BMT patients, clinical GVHD developed after DLI therapy.
Their CDR3 profiles tended to undergo a larger number of transitory
changes than those in the other BMT patients, as illustrated in Figure
4.
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| Fig 4.
Evolution of CDR3 size profiles in patients 4 (DLI +20
months), 9 (DLI +17 months), 10 (DLI +15 months), and 18 (multiple
DLI treatments between +23 and +36 months after BMT), all in whom
GVHD developed, as indicated.
Patterns for 1 BV subfamily are shown for each patient at 6 time points
after BMT.
|
|
Figure 5 summarizes the evolution of CDR3
size patterns in all 19 patients. Overall, spectratypes in BMT
recipients were much more irregular than those in healthy adults and at
least some size classes were completely undetectable within 6 months of
transplant. Also, unlike in healthy adults, in BMT patients some
spectratypes changed considerably over time. However, on the whole, BMT
patients' CDR3 profiles became more normal with increasing time from
BMT (Figure 5). In 1 of the 3 patients who did not achieve molecular complete remission (CR) (Table 2), spectratypes completely normalized between 19 and 24 months post-BMT, whereas some spectratypes remained abnormal by the end of the study more then 36 months after BMT in the
other 2 patients. Hence, these data do not support an association between molecular CR and spectratype normalization.
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| Fig 5.
Summary of CDR3 size spectratyping results from 19 BMT
recipients.
CDR3 size profiles were determined in PBMCs sampled at several time
points after BMT for each patient. All patients underwent DLIs as
indicated  . GvHD developed in 4 patients after DLI.  indicates
that all spectratypes are abnormal;  indicates that some
spectratypes are normal; and  indicates that all spectratypes are
normal. * indicates a patient in whom GVHD developed.
|
|
| |
Discussion |
In all our T-cell-depleted BMT recipients, T-cell repertoires
showed restricted patterns for at least 1 year after BMT. During the
first 3 to 6 months, their BV spectratypes comprised only a very
limited number of size classes. This coincides with the time when BMT
recipients are generally most susceptible to infections. One reason for
the slow and usually incomplete development of complex repertoires is
likely to be the reduced thymic output in adults.3 Roux et
al17,18 discovered that, in the first several months after
transplant, the circulating T-cell repertoire is essentially recruited
from the progeny of the donor lymphocytes cotransplanted with the bone
marrow, and possibly surviving recipient lymphocytes.26
Hence, in patients receiving T-cell-depleted bone marrow,
T-cell-repertoire reconstitution is delayed or incomplete compared
with recipients of unmanipulated BMT. T-cell-repertoire recovery may
depend to a greater extent on the few recipient T cells that may
survive the preconditioning treatment.17,18 This limited
size of the initial pool of T cells in combination with the length of
time it takes them to repopulate the peripheral blood is likely to be a
major factor contributing to the lack of complexity in T-cell
repertoires in the first year after T-cell-depleted BMT. Recent
evidence suggests that, contrary to earlier assumptions, the thymus may
also contribute to immune reconstitution even in adulthood.3 However, the predicted slow tempo of thymic
education in adulthood would similarly contribute to delays in
restoration of a normal spectratype pattern.
There was an overall improvement of spectratypes in most patients after
about 1 year after BMT. This timing coincides with the normalization of
CD8+/CD4+ ratios and with the time when BMT
recipients become less susceptible to opportunistic
infections.4,5 CDR3 size profile normalization was by no
means complete in the majority of patients even after several years and
was highly variable between individual patients and between different
BV families in the same patient. The normalization of all spectratypes
in about a third of patients suggests that complete reconstitution of
T-cell repertoires may be possible. However, this methodology provides
no information about whether there are the same numbers of unique
clones in these patients and their donors within each size class.
In approximately two thirds of the BMT recipients, some BV spectratypes
were still abnormal after several years. Interestingly, despite these
restricted T-cell spectratypes, BMT recipients did not show signs of
impaired immune function, such as increased susceptibility to
infections. There is the possibility that the amount of thymic
selection persisting in adults is variable between individuals.
However, the fact that some BV spectratypes normalize, whereas others
remain abnormal in the same patient, suggests that other factors also
play a part in shaping CDR3 size profiles. Such factors may be
infectious agents encountered after BMT, activation of T cells by
alloantigens, GVHD, and its treatment with immunosuppressive drugs.
Activation of T cells by infectious agents or alloantigens have an
immunosuppressive effect and inhibit the reconstitution of the normal
underlying spectrum of T cells. GVHD can be immunosuppressive, and the
drugs administered to treat GVHD are also toxic or inhibitory to
lymphocytes.4 In agreement with this, none of the 4 patients in this study in whom GVHD developed were among those whose
spectratypes completely normalized. Furthermore, the marked changes in
the CDR3 size profiles observed in these patients over time may be a
result of the combined effect of T-cell responses to the antigens involved in GVHD, and/or the immunosuppressive treatment.
Eighteen patients underwent DLI as a treatment for relapse of their
diseases. As the donor lymphocytes given to BMT recipients during DLI
were samples of a healthy donor T-cell repertoire, they might be
expected to have an immediate normalizing effect on the patients'
T-cell repertoires. However, if the bone marrow was of mixed chimeric
status after transplant, host-versus-graft reactivity could possibly
result in more abnormal spectratypes. Furthermore, in most patients,
DLI gave rise to GVL responses, which eventually resulted in complete
cytogenetic remission. This would require expansion of GVL or GVH
T-cell clones, possibly resulting in a change in the CDR3 size
profiles. Contrary to these predictions, no change in CDR3 size
profiles was detected in half of the BMT recipients shortly after DLI.
This suggests that the number of T cells infused was too small to
immediately change spectratype shapes, and that expansions of clones
mediating GVL or GVH were either too small to be detected or only
occurred at later time points in these patients. Whether these
expansions are demonstrable in terms of spectratype changes will depend
on the relative size of the expansion, the level of expression of the
particular BV family, possibly the level of TCR messenger RNA (mRNA)
per cell, and the position of the CDR3 peak of the expanded clone
within the BV spectratype. A small expansion of a single clone would be
more easily visualized in a relatively underrepresented BV family,
particularly if the CDR3 length was at one extreme of the length
distribution represented in that family. Our data on the sensitivity of
spectratype analysis for the detection of T-cell lines spiked into
PBMCs from a healthy donor suggest that the clone may need to be
represented at a level of 1 in 1000 to 5000 PBMCs to be detectable in
well-represented BV families (and consequently higher relative
frequencies within the CD4+ or CD8+ T-cell
subsets). Clones mediating GVL or GVH may therefore be unrecognizable
if present at lower levels. However, some caution is required in
extrapolation of these results. The thresholds for detection using the
cell lines cannot be directly equated with clonal expanded T cells in
vivo, because TCR mRNA levels are generally higher in activated T cells
than in resting T cells.27 Hence, a lower number of
activated T cells may be detectable against a given background of
resting T cells. Clonal expansion to antigens not involved with GVL or
GVH would be predicted to occur with increasing frequency with
increasing time after DLI, resulting in a gradual normalization of the
spectratype appearance. Although some of the later changes may be
correlated with the expansion of GVL T-cell clones, this methodology is
not able to differentiate these from GVL-independent expansions, or to
differentiate DLI-related spectratype changes from DLI-independent changes.
In only 1 patient did dramatic changes occur in all spectratypes after
DLI (Figure 2B, patient 11), as they changed from normal patterns to
restricted, clonal patterns. Changes in other patients' spectratypes
were limited to a few BVs and were not as clear-cut.
In conclusion, T-cell spectratype analysis in 19 BMT patients sampled
longitudinally for several years after BMT showed that T-cell-repertoire reconstitution differed greatly among patients. There was an overall improvement around 12 months after BMT, but the
time to normalization in the different BV families in each patient was
highly variable. Hence, it would not be possible, as suggested by
Gorski et al,12 to use a few BVs as guideposts for
detection of T-cell impairment. Although immunity residing in donor
memory T cells was lost through T-cell depletion and T-cell
spectratypes did not completely normalize in the majority of our BMT
recipients even after years, this did not seem to affect the patients'
general health.
In most patients, DLI did not result in an immediate change in BV
spectratypes, and later changes could not be unequivocally assigned to
DLI treatment.
| |
Footnotes |
Submitted August 2, 1999; accepted February 9, 2000.
Supported by the Leukemia Research Fund.
Reprints: S. Mackinnon, Department of Haematology, UCL, 98 Chenies Mews, London WC1E 6HX, United Kingdom.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction