From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
TRANSPLANTATION
Direct evidence for new T-cell generation by patients after either T-cell–depleted
or unmodified allogeneic hematopoietic stem cell transplantations
Sharon R. Lewin, Glenn Heller, Linqi Zhang, Elaine Rodrigues, Eva Skulsky, Marcel R. M. van den Brink,
Trudy N. Small, Nancy A. Kernan, Richard J. O’Reilly, David D. Ho, and James W. Young
Successful allogeneic hematopoietic stem
cell transplantation (HSCT) requires reconstitution of normal T-cell immunity.
Recipient thymic activity, biologic features of the allograft, and preparative
regimens all contribute to immune reconstitution. We evaluated circulating T-cell
phenotypes and T-cell receptor rearrangement excision circles (TRECs) in 331
blood samples from 158 patients who had
undergone allogeneic HSCTs. All patients
had received myeloablative conditioning
regimens and were full donor chimeras in
remission. Younger patients exhibited
more rapid recovery and higher TRECs
(P ⴝ .02). Recipients of T-cell–depleted
allografts initially had lower TRECs than
unmodified allograft recipients (P < .01),
but the difference abated beyond 9
months. TREC level disparities did not
achieve significance among adults with
respect to type of allograft. Measurable,
albeit low, TREC values correlated
strongly with severe opportunistic infections (P < .01). This finding was most
notable during the first 6 months after
transplantation, when patients are at
greatest risk but before cytofluorography
can detect circulating CD45RAⴙ T cells.
Low TRECs also correlated strongly with
extensive chronic graft-versus-host disease (P < .01). Recipients of all ages of
either unmodified or T-cell–depleted allografts therefore actively generate new T
cells. This generation is most notable
among adult recipients of T-cell–depleted
allografts, most of whom had also received antithymocyte globulin for rejection prophylaxis. Low TREC values are
significantly associated with morbidity
and mortality after transplantation. T-cell
neogenesis, appropriate to age but delayed in adult recipients of T-cell–
depleted allografts, justifies interventions to hasten this process and to stimulate desirable cellular immune responses.
(Blood. 2002;100:2235-2242)
© 2002 by The American Society of Hematology
Introduction
Impaired reconstitution of T-cell–mediated immunity is a major
cause of morbidity and mortality after allogeneic hematopoietic
stem cell transplantation (allogeneic HSCT). T-cell regeneration
after allogeneic HSCT can occur by several mechanisms, including
thymic-dependent and thymic-independent pathways. Investigators
have usually followed the recovery of specific T-cell subsets, such
as CD4⫹, CD8⫹, and CD45RA⫹, to monitor immune reconstitution
after allogeneic HSCT, as well as high-dose chemotherapy or
infections such as HIV.1-7 Freedom from opportunistic infections
(OIs) correlates strongly with numerical recovery of these and
other phenotypically distinct lymphocyte subsets.1,2,6,7 Even when
using rare event analysis for cytofluorographic assessments of
circulating CD45RA⫹ T cells, however, the numbers of circulating
T cells or T-cell subsets are just too low for detection during the
early months after transplantation when patients are at greatest risk
of complications from immune deficiency.
As progenitor T cells undergo T-cell receptor (TCR) gene
rearrangement in the thymus, chromosomal sequences are excised
to produce episomal DNA byproducts termed TCR rearrangement
excisional circles (TRECs). TRECs in circulating T lymphocytes
therefore mark these cells as recent thymic emigrants (RTEs).8
TRECs persist but decrease with age. TRECs can also increase
after effective therapy for HIV infection or after thymic transplantation.8-12 Because TRECs are nonreplicating episomal DNA,
TRECs are lost only by cell death, dilution through cell division, or
both.13 TRECs therefore represent the composite production,
proliferation, and death of RTEs.9,10,14,15
Our study was designed to determine the mechanism and kinetics of
T-cell regeneration after allogeneic HSCT, especially in a series dominated by older adult recipients of T-cell–depleted allografts. This group
has long posed a quandary regarding immune reconstitution, as to
whether there are host limitations to T-cell regeneration and/or graft
limitations from the quantity and quality of transplanted T-cell precursors. TRECs offer a means of directly addressing these important
unknowns. TREC measurements are also potentially more sensitive
than immunophenotypic monitoring, especially when circulating T cells
are numerically low or undetectable by this method early after transplantation. We therefore evaluated the relation of TRECs to other host and
From the Aaron Diamond AIDS Research Center, The Rockefeller University;
and the Department of Epidemiology and Biostatistics, Allogeneic Bone
Marrow Transplantation Service, Departments of Pediatrics and Medicine,
Memorial Sloan-Kettering Cancer Center, New York, NY.
and Medical Research Council of Australia and the Ian Potter Foundation.
Submitted October 24, 2001; accepted May 13, 2002.
Supported by the Irene Diamond Fund (S.R.L., L.Z., E.S., D.D.H.); grants R01
AT 46964 (L.Z.) from the National Institute of Allergy and Infectious Diseases,
National Institutes of Health; P01 CA 23766 (G.H., M.R.M.vdB., T.N.S., N.A.K.,
R.J.O., J.W.Y.) and R01 CA 83070 (J.W.Y.) from the National Cancer Institute,
National Institutes of Health; and LLS 6124-99 (J.W.Y.) from The Leukemia and
Lymphoma Society of America. S.R.L. is also supported by the National Health
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
Presented in part in abstract form at the 42nd Annual Meeting of the American
Society of Hematology, San Francisco, CA, December 2000.
Correspondence: Sharon R. Lewin, Department of Microbiology and
Immunology, University of Melbourne, Royal Parade, Parkville, Victoria, 3052,
Australia; e-mail: [email protected].
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.
© 2002 by The American Society of Hematology
2235
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2236
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
LEWIN et al
transplant factors such as donor and recipient age, type of graft, degree
of HLAmatching, clinically significant graft-versus-host disease (GvHD),
and radiation dose. We also assessed the physiologic significance of
measured TRECs with respect to the presence or absence of severe OIs.
Patients, materials, and methods
Patient, donor, and transplant characteristics
We obtained 5-mL peripheral blood samples from consecutive, consenting
patients who were in the hospital at least 1 month after transplantation and
engrafted, or who were returning for regularly scheduled outpatient
followup after allogeneic bone marrow or granulocyte colony-stimulating
factor (G-CSF)–elicited peripheral blood stem cell transplants (Table 1).
Regardless of whether the regimen included total body irradiation or not, all
patients received myeloablative conditioning. This series does not include
any adult patients treated with nonmyeloablative preparative regimens for
allogeneic HSCT. Most of the patients received transplants for lymphohematopoietic malignancies. All patients who received unmodified allografts
also received standard pharmacologic prophylaxis against GvHD with
cyclosporin or tacrolimus plus methotrexate. T-cell–depleted allograft
recipients received no additional GvHD prophylaxis, but 106 (84%) of 127
T-cell–depleted allograft recipients received a brief course of antithymocyte
globulin (ATG) and steroids in the peritransplantation period to prevent
graft rejection.16-20 HLA allelic mismatching was based on DNA nonidentity at any one of the 6, codominant HLA-A, -B, or -DR␤1 loci. All patients
were in hematologic and/or cytogenetic remission and were fully donor
engrafted at the time of sampling. Patient sampling was conducted in
accordance with Institutional Review Board sanctioned protocols.
TREC quantification
Peripheral blood mononuclear cells (PBMCs) were isolated from the
interface after centrifugation of approximately 5 mL whole venous blood on
Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ). Cells
were frozen in a pellet at ⫺80°C until DNA extraction. Frozen cell samples
were thawed on ice, and genomic DNA was extracted by using a QIAamp
DNA Blood Mini Kit (no. 51106; QIAgen, Valencia, CA) according to the
manufacturer’s instructions. The DNA from each processed sample was
then frozen at ⫺80°C until tested for TRECs. TRECs and the corresponding
number of cell equivalents were quantified by using real-time polymerase
chain reaction (PCR).9 A molecular beacon, designed to recognize a region
upstream from the signal joint, was included in the reaction mixture
throughout PCR to serve as a real-time detector for the amplified product.
This beacon was specifically designed to have a hairpin structure, with a
6-bp stem and a 26-nucleotide target-recognition loop, plus a fluorophore
[FAM (6-carboxyfluorescein)] and quencher [DABCYL (4-dimethylaminophenlazo benzoic acid)] in close proximity at the 2 ends of the
oligonucleotide.
The PCR reaction was conducted in a spectrofluorometric thermal
cycler (ABI PRISM 7700; Applied Biosystems, Foster City, CA) that
monitors changes in the fluorescence spectrum of each reaction tube during
the annealing phase, while simultaneously carrying out programmed
temperature cycles. The cycle number during PCR that yields a fluorescent
intensity significantly above the background is designated as the threshold
cycle (CT). The CT is directly proportional to the log of the copy number of
the target sequence in the input DNA. We have previously reported that the
assay is efficient (⬍ 4 hours), specific, sensitive (100 copies), dynamic
(7-log range), and accurate (10% coefficient of variance).9
Immunologic and clinical monitoring
Immunophenotyping used fluorochrome-conjugated antibodies to CD3,
CD4, CD8, CD45, and CD45RA, after which cells were analyzed on a
FACScan (BD Pharmingen, San Diego, CA).2 Cytomegaloviremia (CMV
antigenemia) was detected by positive immunofluorescent staining of the
pp65 antigen (Chemicon, Temecula, CA) in circulating leukocytes. CMV
disease was defined by identification of CMV in the liver, gut, lungs, and/or
other tissue site, together with circulating CMV antigenemia. Table 2 lists
additional severe OIs and their incidences. Clinically significant acute
GvHD included overall grade II to IV disease,21 and clinically significant
chronic GvHD comprised extensive but not limited disease.22
Data and statistical analyses
TRECs were quantified per 106 input PBMCs. All values less than 100 were
normalized to 100 for purposes of further calculations and analysis, because
the valid detection limit of the assay was 100 TRECs/106 PBMCs. Almost
all patients had simultaneous lymphocyte phenotyping as above at the time
of TREC sampling. Excluding those patients who had undetectable CD3⫹ T
cells (TREC values relative to CD3⫹ T cells could not be calculated when
CD3⫹ T-cell denominators were zero) and the rare patients whose CD3⫹ T
cells were not simultaneously assayed with TREC sampling, TRECs were
also calculated per 106 CD3⫹ T cells [TRECs per million CD3⫹ T
cells ⫽ (TRECs per 106 PBMCs)/(percent CD3⫹ T cells/100)]. These calculations and analyses generated similar curves and P values to those of TRECs
per 106 PBMCs, so these findings have not been separately illustrated.
Stratified permutation tests were performed using the Wilcoxon rank
sum statistic to determine the level of association between TRECs and
prognostic factors. The stratification variable was the time after transplantation at which the TREC value was measured. The test statistic was stratified
into time intervals so that TREC (rank) values could be compared at
comparable time periods. Although there were multiple observations for
more than half the patients, the permutation procedure was performed at the
patient level to maintain the correlation structure induced by multiple
observations. For the purpose of this stratified analysis, the time since
transplantation was divided into 7 groups: 1 to 3 months, 3 to 6 months, 6 to
9 months, 9 to 12 months, 12 to 24 months, 24 to 48 months, and more than
48 months.
To depict the graphic relationship between TRECs and the prognostic
factors studied, controlling for the elapsed time since transplantation, a
nonparametric smoothing procedure was performed.23 The figures depict all
data points, but the connecting curves have been truncated for groups in
which data became too sparse to allow a valid fit.
The amount of variation in TRECs per 106 PBMCs predicted by the
percentage of circulating CD4⫹ CD45RA⫹ T cells and the time since
transplantation were summarized by using the goodness of fit measure r2.
This statistic is based on a nonparametric generalized additive model24 that
relates these 3 variables and is similar to the r2 statistic used to summarize
the goodness of fit in linear regression models. The statistic varies between
zero and 1, with high r2 values indicating that an accurate prediction of an
individual TREC outcome can be obtained through knowledge of an
individual’s percentage of circulating CD4⫹ CD45RA⫹ T cells and the time
since transplantation that it was recorded.
Results
Younger patients exhibit more robust generation of new T cells
TRECs were significantly higher in those patients who were
younger than 19 years at the time of allogeneic HSCT, compared
with those 19 years and older (P ⫽ .02; Figure 1). Even adults,
however, had detectable TRECs after myeloablative and lymphoablative conditioning for allogeneic HSCT. There were too few
pediatric recipients of unrelated adult donor grafts and insufficient
disparity in the overall group between recipient and donor age to
detect an independent effect of donor age on TRECs.
Active generation of new T cells occurs after either
unmodified or T-cell–depleted allogeneic HSCT
We compared circulating TRECs in recipients of T-cell–depleted
and unmodified (T-cell replete) allografts. Of the T-cell–depleted
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
THYMIC ACTIVITY AFTER HEMATOPOIETIC ALLOGRAFTS
2237
Table 1. Patient, donor, and transplant characteristics
No. patients/no. samples
Unmodified transplant
T-cell–depleted transplant
Total
31/56
127/275
158/331
13/19
87/189
100/208
2/2
11/27
13/29
16/35
29/59
45/94
1-93
1-138
—
Donor
Related, HLA identical, no. patients/no. samples
Related, HLA nonidentical, no. patients/no. samples
Unrelated, HLA identical or nonidentical, no. patients/no. samples
Time of sample collection after transplantation, range in mo
Age
Range, y
2.2-52.3
2.9-61.5
—
Younger than 19 y, (%)*
9 (29)
15 (12)
24 (15)
19 y or older, (%)
22 (71)
112 (88)
134 (85)
35 y or older, (%)
9 (29)
72 (56)
81 (58)
40 y or older, (%)
4 (13)
62 (49)
66 (42)
Younger than 19 y
0
1
19 y or older
0
0
Younger than 19 y
0
1
19 y or older
0
0
Younger than 19 y
0
0
19 y or older
1
1
Younger than 19 y
9
13
19 y or older
21
111
Younger than 19 y
5/10
1/3
19 y or older
3/4
4/5
Younger than 19 y
0/0
2/7
19 y or older
0/0
0/0
3/9
0/0
10/20
0/0
Younger than 19 y
0/0
3/5
19 y or older
6/7
27/58
Younger than 19 y
0/0
0/0
19 y or older
1/1
11/24
Younger than 19 y
1/2
9/24
19 y or older
2/3
70/149
Primary disease for which allogeneic HSCT performed (no. patients)
Juvenile rheumatoid arthritis, severe
1
Thalassemia major
1
Aplastic anemia
2
Lymphohematopoietic malignancies† or myelodysplastic syndromes
154
Total body irradiation, dose (cGy), no. patients/no. samples‡
0
13/22
450
2/7
1350
Younger than 19 y
19 y or older
13/29
1375
36/70
1410
12/25
1500
82/178
ATG graft rejection prophylaxis
Yes (%)*
0 (0)
105 (83)
105
31 (100)
22 (17)
53
9/21
14/40
23/35
93/202
Younger than 19 y
0/0
1/1
19 y or older
0/0
18/34
No (%)
TRECs sampled after donor leukocyte infusions (DLI), no. patients/no. samples
No DLI
Younger than 19 y
19 y or older
139/296
Prior DLI
19/35
*Percentage calculated relative to the total number of patients in the column category.
†Includes patients allografted as treatment for acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, non-Hodgkin lymphoma, and a
single case of multiple myeloma.
‡All preparative regimens were myeloablative and included either radiation and chemotherapy or chemotherapy alone.
allograft recipients, 83%, who were all adults, had received ATG
for the prevention of graft rejection. Recipients of unmodified
allografts were treated with standard pharmacologic prophylaxis
against GvHD, using methotrexate and either cyclosporine or
tacrolimus. TRECs were initially more numerous in the recipients
of unmodified allografts (P ⬍ .01; Figure 2), but this difference
abated beyond 9 months after transplantation. Furthermore, when
pediatric patients were excluded and only adults aged 19 years and
older at the time of transplantation were analyzed, the relationship
between TRECs and type of graft (unmodified versus T-cell
depleted), controlling for elapsed time from transplantation, diminished (P ⫽ .07 instead of P ⬍ .01). Adult recipients of either type
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2238
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
LEWIN et al
Table 2. Opportunistic infections in recipients of allogeneic
hematopoietic stem cell transplants
Type of severe or life-threatening OI
N
Cytomegaloviremia, chronic/refractory
4
Cytomegalovirus (CMV) end-organ disease (pneumonitis, 2; retinitis, 1)
3
Epstein Barr virus lymphoproliferative disorder
20
Fungal pneumonia
7
Fungemia/fungal sepsis or disseminated fungal disease
8
Herpes simplex virus, disseminated
3
JC polyomavirus (progressive multifocal leukoencephalopathy)
1
Legionella pneumonia
1
Listeria monocytogenes sepsis
2
Mycobacterial (nontuberculous) infection
6
Mycobacterium tuberculosis
3
Nocardia sepsis or disseminated disease
3
Pneumocystic carinii pneumonia (PCP)
2
Respiratory syncytial virus (RSV) pneumonia
2
Toxoplasmosis gondii
2
Viral hemorrhagic cystitis
1
Opportunistic infection (OI) occurred in 58 allogeneic HSCT recipients. Eight
recipients had 2 opportunistic infections over the course of the study. One individual
had 3 opportunistic infections.
of transplant recovered TREC values during the second year after
allogeneic HSCT that were as high or higher than those measured
in historical healthy controls9 (median TRECs per 106 PBMCs in
adults aged 19-62 years were 3620 in healthy donors,9 1087 in
patients between 9 and 12 months after transplantation, and 10 853
in patients between 12 and 24 months after transplantation). There
were unfortunately too few data points from patients younger than
19 years to make similarly valid comparisons.
Naive T-cell phenotype predicts but does not account for all of
the newly generated T cells after allogeneic HSCTs
The only naive T-cell phenotype followed in this series was
coexpression of the CD45RA isoform by CD4⫹ T cells. The
absolute number and the percentage of CD4⫹ CD45RA⫹ T cells
were evaluated, and the percentage values were more predictive of
the TREC values. Eighty percent of the CD4⫹ CD45RA⫹ values
were 5% or less, and 1% was the median. We therefore compared
TRECs between samples dichotomized by the median of 1%
CD4⫹CD45RA⫹ T cells. Higher TRECs indeed predominated in
those samples with more than 1% CD4⫹CD45RA⫹ T cells
(P ⬍ .01; Figure 3). The variation in TRECs attributable to the time
after transplantation and the observed percentage of circulating
Figure 1. TRECs and patient age at time of transplantation. Circulating TRECs
per 106 PBMCs were measured and plotted (y-axis) for patients who were younger
than 19 years (f, solid line) versus patients who were 19 years or older (E, dashed
line) at the time of transplantation, as a function of time from transplantation (x-axis).
The difference between the 2 curves is significant at P ⫽ .02. Note that the lower limit
of detection for TRECs was 100 TRECs/106 PBMCs in the assay used.
Figure 2. TRECs after T-cell–depleted or unmodified (T-cell replete) allogeneic
hematopoietic stem cell transplantations. Circulating TRECs per 106 PBMCs
were measured and plotted (y-axis) for patients who received unmodified (f, solid
line) versus T-cell–depleted (E, dashed line) allogeneic HSCTs, as a function of time
from transplantation (x-axis). The difference between the 2 curves is significant at
P ⬍ .01, but analysis by quartiles (not shown) indicated that the differences between
the 2 groups diminished beyond 9 months. Note that the lower limit of detection for
TRECs was 100 TRECs/106 PBMCs in the assay used.
CD4⫹CD45RA⫹ T cells was only 0.45, however. Differences in the
proportion of T cells expressing the naive, CD4⫹ CD45RA⫹
surface phenotype therefore could not predict more than half (55%)
of the variation in measured TRECs (r2 derived from the generalized additive regression model). The percentage of circulating
CD4⫹ CD45RA⫹ T cells exerted a 7.5-fold greater contribution to
the predicted TRECs in samples obtained more than 9 months after
transplantation, compared with those obtained earlier.
Degree of genotypic HLA identity does not affect the
generation of new T cells after allogeneic HSCT
We compared TREC values from patients who had received
allogeneic HSCTs from genotypically HLA-identical, related donors with those who had received allogeneic HSCTs from either
HLA-nonidentical, related donors or any unrelated donors (Table
1). Unrelated donors, even if HLA-identical by DNA, are by
definition phenotypically but not genotypically identical to their
recipients because of different parentage. There was no effect on
TRECs exerted by the presence or absence of genotypic HLA
identity between host-donor pairs (P ⫽ .79; Figure 4).
Figure 3. The relationship between TRECs and the percentage of circulating
CD4ⴙ CD45RAⴙ T lymphocytes. TRECs per 106 PBMCs were measured and
plotted (y-axis) for samples that had greater or less than 1% circulating CD4⫹
CD45RA⫹ lymphocytes (ⱕ 1%, f, solid line versus ⬎ 1%, E, dashed line), as a
function of time from transplantation (x-axis). The difference between the 2 curves is
significant at P ⬍ .01. Differences in the proportion of T cells expressing the naive,
CD4⫹ CD45RA⫹ surface phenotype, however, could not account for 55% of the
variation in measured TRECs (r2 derived from nonparametric regression model by
using same data depicted). Note that the lower limit of detection for TRECs was 100
TRECs/106 PBMCs in the assay used.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
THYMIC ACTIVITY AFTER HEMATOPOIETIC ALLOGRAFTS
2239
Figure 4. The effect of HLA identity on TREC recovery after transplantation.
TRECs per 106 PBMCs were measured and plotted (y-axis) for samples from patients
who were either HLA genotypically identical with their donor (f, solid line) or not (E,
dashed line), as a function of time from transplantation. The latter group included all
who had received related but HLA nonidentical allografts or who had received
allografts from any unrelated donor (E, dashed line). On the basis of this dichotomization, there was no difference in TRECs between the 2 groups (P ⫽ .79). Note that the
lower limit of detection for TRECs was 100 TRECs/106 PBMCs in the assay used.
Quantification of TRECs is physiologically significant
after allogeneic HSCT
TREC values were compared between patients who did or did not
develop clinically significant GvHD. We did not identify an
association between TRECs and acute GvHD. In contrast, the
presence of extensive chronic GvHD was strongly associated with
low TRECs (P ⬍ .01; Figure 5). This association is despite the fact
that only 10 patients in the overall series had extensive chronic
GvHD, 2 of whom had inactive disease at the time of sampling.
One case of extensive chronic GvHD had occurred after donor
leukocyte infusions, 4 had occurred after unrelated allografts, and
one had occurred after a mismatched related allograft. Nine of the
10 cases were in adults.
TRECs were also compared between patients who did or did not
develop severe OIs (Table 2). Of the 58 individuals who developed
a severe or life-threatening OI, 8 individuals had more than one OI;
but analyses were based only on whether an individual patient did
or did not have a history of any severe or life-threatening OI. Both
children (younger than 19 years) and adults (19 years and older)
who developed higher numbers of TRECs over time from transplantation were less likely to suffer from severe OIs. Conversely, low
TRECs were associated with the presence of severe OIs (P ⬍ .01;
Figure 6A). The relationship between OI and TREC values
Figure 5. TREC recovery in the presence or absence of extensive chronic
GvHD. TRECs per 106 PBMCs were measured and plotted (y-axis) for samples from
patients who had developed extensive chronic GvHD (n ⫽ 10; E, dashed line) versus
those who had developed either limited chronic GvHD or none at all (f, solid line), as
a function of time from transplantation. Three patients had more than one TREC
measurement, giving a total of 18 samples from this group of 10 patients. There was a
highly significant difference between the 2 groups (P ⬍ .01). Note that the lower limit
of detection for TRECs was 100 TRECs/106 PBMCs in the assay used.
Figure 6. Low TRECs are associated with the development of OIs. TRECs per
106 PBMCs were measured and plotted (y-axis) for patients who had developed
severe OIs (E, dashed line) or not (f, solid line) as a function of the time from
transplantation. All severe OIs are represented in A (P ⱕ .01; CMV is included only for
chronic or refractory viremia; see also Table 2). (B) This panel includes any CMV
antigenemia and/or end-organ CMV disease (P ⫽ .05). Note that the lower limit of
detection for TRECs was 100 TRECs/106 PBMCs in the assay used.
remained strong (P ⬍ .01) after adjustment for other factors (eg,
age, CD4⫹ T-cell count, CD4⫹CD45RA⫹ T-cell count, type of
transplant, and presence or absence of chronic GvHD).
Among the various OIs seen in this series, the most common
was reactivation of CMV detected in circulating leukocytes (CMV
antigenemia). Actual CMV infection or disease also occurred, as
did chronic or refractory CMV antigenemia, but these were
included in the above analysis in Figure 6A. These events were
relatively rare within the overall group with reactivated CMV,
given the current clinical practice of routine surveillance for earlier
detection and preemptive antiviral therapy of CMV antigenemia.
The occurrence of CMV antigenemia and/or its treatment with
antiviral drugs was nevertheless also associated with low TREC
values (P ⫽ .05; Figure 6B).
The percentages of circulating CD4⫹ CD45RA⫹ T cells were
also compared between patients who did or did not develop OIs
after transplantation. Lower percentages of circulating T cells
expressing this naive phenotype correlated strongly with the
presence of severe OIs (P ⬍ .01) and slightly less so with CMV
antigenemia or disease (P ⫽ .06). Again, adjustments for other
potentially contributing factors did not affect the association
between OIs and the percentage of circulating CD4⫹ CD45RA⫹
T cells.
Discussion
Successful reconstitution of T-cell–mediated immunity substantially reduces morbidity and mortality after allogeneic HSCT. We
therefore used TRECs to investigate potential mechanisms of
immune reconstitution after allogeneic HSCT. In a series dominated by adult recipients of T-cell–depleted allografts, most of
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2240
LEWIN et al
whom also received total body irradiation and ATG, active T-cell
neogenesis did occur; TRECs recovered at least age appropriate
levels by the second year after transplantation. Measurable deficiencies in TREC values significantly correlated with clinical morbidity
and mortality, especially during the early posttransplantation
period of greatest risk for severe OIs. This finding was in contrast to
the detection of circulating CD4⫹ CD45RA⫹ cells, often used as an
indicator of new T-cell generation, but whose recovery actually
heralds a decreasing risk of severe OIs.1,2,6
Hosts have no measurable T cells after myeloablative preparative regimens for allogeneic HSCT. Only newly synthesized T cells
or proliferation of T cells transferred with the allograft could
account for any real expansion of T cells. Most of the patients in
this series, however, received T-cell–depleted allografts comprising
fewer than 105 clonable T cells per kilogram at the outset.25 This
limits the initial pool for T-cell expansion and dilution of TRECs,
independent of T-cell neogenesis. It also contrasts with the higher
numbers of residual T cells after other depletion methods or the
approximate dose of 107 clonable T cells per kilogram administered
to unmodified transplant recipients. Given that TRECs are undetectable at the time of transplantation, alterations in T-cell proliferation
alone would not explain changes in the absolute numbers of
TREC⫹ cells. The observed increases in TREC levels must
therefore have been associated with the generation of new donorderived T cells, notwithstanding the influence of T-cell activation
and proliferation on TREC measurements after initial T-cell
repopulation.15
Other investigators have measured TRECs to evaluate immune
recovery after autologous26 or allogeneic HSCTs,27-30 but most of
these studies have focused on children and young adults. Recent
work has demonstrated a reduction in T-cell proliferation in the
steady state, contrasted with increased proliferation in response to
chronic antigenic stimulation associated with infection or GvHD
after allogeneic HSCT.15 This research highlights the influence that
clinical status can have on measured TRECs and reinforces the
paradigm that TREC levels represent new T-cell production
counterbalanced by the competing factors of either T-cell death or
T-cell proliferation that dilutes TRECs among the progeny, or both.
The series reported here is unique, however, in its large number
of samples; a 4:1 preponderance of T-cell–depleted over unmodified allograft recipients; the exclusive use of myeloablative conditioning regimens for transplantation of mostly lymphohematopoietic malignancies; and the presence of full donor T-cell chimerism
in all patients at the time of sampling. There were also several
variables that we anticipated might have negatively biased the
results but did not. For example, approximately half the adults were
35 years or older at transplantation. Most adults also received
T-cell–depleted allografts, and 83% received prophylaxis against
graft rejection with ATG that could have destroyed RTEs because
of its persistence in the circulation. Despite this, adult patients
recovered age-appropriate and possibly even higher levels of
TREC⫹ cells than age-matched historical healthy control subjects9
during the second year after transplantation.
There was a strong association between TRECs and the
percentage of circulating CD4⫹ CD45RA⫹ lymphocytes, but this
association was almost entirely limited to samples obtained beyond
9 months after transplantation. Use of this phenotype as a surrogate
approximation of T-cell neogenesis can therefore confound accurate estimates of thymic activity. There were in fact patients early
after transplantation who had detectable TRECs but no detectable
circulating T cells by flow cytometry. The subsequent recovery of
CD4⫹ CD45RA⫹ T cells heralds the time after transplantation
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
when the risk of OI markedly decreases,2,6 rather than providing a
discriminating correlate during the period of greatest risk.
Other variables beyond coexpression of CD4 and CD45RA,
either not observed or not assessed in this study, could also have
contributed to measured TRECs. Possibilities include T cells
expressing additional naive phenotypes such as CD8⫹CD103⫹31
and/or CD8⫹CD45RA⫹; additional surface markers such as CD62L,
CD11a, CD27, and CD2832; or Ki67, indicating active proliferation
and dilution of TRECs among the T-cell progeny.15 The CD4⫹
CD45RA⫹ phenotype could also overestimate T-cell neogenesis, as
T cells expressing the CD45RA isoform may expand in the absence
of specific antigenic stimulation and maintain CD45RA expression.33 CD45RA⫹ T cells may also be phenotypic revertants from
CD45RO⫹ memory T cells.34
Higher TREC values were indeed associated with a decreased
incidence of CMV antigenemia and a decreased incidence of severe
OIs overall. Conversely, lower TREC values were associated with a
significantly higher incidence of these complications. We considered a number of other factors (eg, age, CD4⫹ T-cell count, CD4⫹
CD45RA⫹ T-cell count, type of transplant, and presence or absence
of chronic GvHD) that might affect the relationship between severe
OI and TREC values, but none of these factors independently
influenced this association. On the basis of these data alone, we
cannot distinguish whether the lower TREC levels result from
severe OIs or alternatively reflect graft-host interactions independent of infection that decrease TRECs and increase infection risk in
the posttransplantation period.
That TRECs recovered to levels as high or higher than historical
healthy controls in adult patients could indicate rapid entry of
TREC⫹ naive T cells into a virtually empty T-cell pool, which
would markedly increase the TREC concentration without a
meaningful increase in the absolute TREC number. Alternatively, a
“supranormal” TREC concentration may reflect both robust T-cell
neogenesis and a concomitant reduction in T-cell proliferation over
time.15 Although restoration of TCR diversity was not specifically
evaluated in this series, such recovery of the T-cell repertoire has
been associated with the ability to respond to vaccination6 and a
lower susceptibility to OIs.2
Despite the low incidence of extensive chronic GvHD in this
study, there was a strong association between this posttransplantation complication and low TRECs as previously reported.28 Extensive chronic GvHD is deleterious to clinical immune competence,22
and mouse data have demonstrated GvHD-induced injury to host
lymphoid organs required for reconstitution of T-cell immunity.35
In addition, the chronic T-cell stimulation and turnover associated
with extensive chronic GvHD could decrease TREC numbers.15
In contrast, we did not ascertain an independent effect of
radiation dose used in the preparative regimen (1350-1500 cGy
range), which we had hypothesized might affect host lymphoid
niches for regenerating T cells. In the absence of lymphoid sites
where new T cells can encounter antigen and undergo proliferation and dilution of TRECs, values may remain misleadingly
elevated. We did at least confirm in vitro, however, that
engrafted T cells have the capacity to divide and decrease their
TREC content in response to stimulation with a polyclonal
mitogen (data not shown).
Comparisons between T-cell–depleted and unmodified allograft
recipients demonstrated a more significant increase in TREC levels
after unmodified allografts, but only in the first 9 months after
transplantation. When adults only were examined, however, the
relationship between TRECs and type of transplant diminished
(P ⫽ .07 versus P ⫽ .02). Age may therefore have contributed only
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
to the early differences between type of transplant and TRECs,
because of the disproportionate representation of children in the
unmodified transplant group and the preponderance of adults in the
T-cell–depleted transplant group. This finding is consistent with
prior studies from our institution in which children have always
reconstituted normal immunity earlier than adults regardless of
type of donor or transplant.1,2 There were too few pediatric
recipients of unrelated adult donor grafts and insufficient disparity
in the overall group between recipient and donor age to detect an
independent effect of donor age on TRECs.
Because pediatric patients reconstitute normal cellular immunity and achieve a lower risk status for complications earlier than
adults, most return to their referring physicians for followup sooner
than adults. Accordingly, there were very few pediatric samples
after 24 months from transplantation, given the manner in which
we sampled consecutive, consenting patients returning for regular
followup at this center. When we analyzed each parameter over the
entire sampling period but included only adults, almost identical
curves and similar P values were obtained as for the whole
population (not shown). There was one expected exception, which
was the relationship between TRECs and type of transplant that
was influenced by the inclusion of children.
Donor leukocyte infusions (DLIs) administered in this series
were predominantly for molecular or cytogenetic relapse of CML
and were dosed according to the content of bulk CD3⫹ T cells.
Only 12% of the patients in this series received DLI, however, and
these patients only accounted for 10% of the total samples. We
detected no influence on TRECs, either because the numbers were
too small or because DLI really exerted no effect.
Finally, we considered whether late sampling after transplantation could have selected for patients surviving long enough to
generate higher TRECs. This is unlikely, however, because in all
situations in which substantive differences in TRECs were
THYMIC ACTIVITY AFTER HEMATOPOIETIC ALLOGRAFTS
2241
noted, the differences occurred within the first 9 to 12 months
rather than later.
In conclusion, these results demonstrate that adult recipients of
T-cell–depleted allografts, most of whom also received total body
irradiation and ATG, can actively generate new T cells based on
increasing TREC levels. Recipient hosts of all ages did in fact
increase their TREC values after transplantation of either unmodified or T-cell–depleted allografts. Low TRECs and low percentages
of circulating CD4⫹CD45RA⫹ T cells were strongly associated
with an increased incidence of severe OIs. TRECs were measurable, however, even before flow cytometry could detect circulating
CD4⫹CD45RA⫹ T cells. TREC values are therefore an important
correlate for severe OIs during the period of greatest risk, and their
predictive value in this setting warrants further prospective evaluation. Extensive chronic GvHD, although limited in incidence in this
study, also had a strong negative association with TRECs, consistent with GvHD-mediated destruction of lymphoid niches for the
regeneration of T-cell immunity35 or with chronic T-cell stimulation
and turnover,15 or a combination of both processes.
Select individuals with low TREC values may therefore benefit
from targeted interventions to hasten new T-cell production or from
preemptive antimicrobial therapy against severe OIs when they are
at greatest risk. The active generation of new donor-derived T cells
also justifies measures to enhance acquired cellular immunity
against residual disease or opportunistic microbes. These hypotheses warrant prospective evaluation in clinical trials.
Acknowledgments
We thank Frieda Toomasi, RN, the nurses, and attending physicians
of the Allogeneic Bone Marrow Transplantation Service and Drs
Jan Baggers and Gudrun Ratzinger, all at MSKCC, for assistance
with sample procurement and processing.
References
1. Keever CA, Small TN, Flomenberg N, et al. Immune reconstitution following bone marrow transplantation: comparison of recipients of T-cell depleted marrow with recipients of conventional
marrow grafts. Blood. 1989;73:1340-1350.
2. Small TN, Papadopoulos EB, Boulad F, et al.
Comparison of immune reconstitution after unrelated and related T-cell-depleted bone marrow
transplantation: effect of patient age and donor
leukocyte infusions. Blood. 1999;93:467-480.
3. Mackall CL, Granger L, Sheard MA, Cepeda R,
Gress RE. T-cell regeneration after bone marrow
transplantation: differential CD45 isoform expression on thymic-derived versus thymic-independent progeny. Blood. 1993;82:2585-2594.
4. Mackall CL, Fleisher TA, Brown MR, et al. Age,
thymopoiesis, and CD4⫹ T-lymphocyte regeneration after intensive chemotherapy. N Engl
J Med. 1995;332:143-149.
5. Autran B, Carcelain G, Li TS, et al. Positive effects of combined antiretroviral therapy on CD4⫹
T cell homeostasis and function in advanced HIV
disease. Science. 1997;277:112-116.
6. Roux E, Dumont-Girard F, Starobinski M, et al.
Recovery of immune reactivity after T-celldepleted bone marrow transplantation depends
on thymic activity. Blood. 2000;96:2299-2303.
7. Ledergerber B, Mocroft A, Reiss P, et al. Discontinuation of secondary prophylaxis against Pneumocystis carinii pneumonia in patients with HIV
infection who have a response to antiretroviral
therapy. Eight European Study Groups. N Engl
J Med. 2001;344:168-174.
8. Douek DC, McFarland RD, Keiser PH, et al.
Changes in thymic function with age and during
the treatment of HIV infection. Nature. 1998;396:
690-695.
9. Zhang L, Lewin SR, Markowitz M, et al. Measuring recent thymic emigrants in blood of normal
and HIV-1-infected individuals before and after
effective therapy. J Exp Med. 1999;190:725-732.
10. Jamieson BD, Douek DC, Killian S, et al. Generation of functional thymocytes in the human adult.
Immunity. 1999;10:569-575.
11. Poulin J-F, Viswanathan MN, Harris JM, et al. Direct evidence for thymic function in adult humans.
J Exp Med. 1999;190:479-486.
12. Markert ML, Boeck A, Hale LP, et al. Transplantation of thymus tissue in complete DiGeorge syndrome. N Engl J Med. 1999;341:1180-1189.
13. Livak F, Schatz DG. T-cell receptor alpha locus
V(D)J recombination by-products are abundant in
thymocytes and mature T cells. Mol Cell Biol.
1996;16:609-618.
14. Hazenberg MD, Otto SA, Cohen Stuart JW, et al.
Increased cell division but not thymic dysfunction
rapidly affects the T-cell receptor excision circle
content of the naive T cell population in HIV-1 infection. Nat Med. 2000;6:1036-1042.
15. Hazenberg MD, Otto SA, de Pauw ES, et al. Tcell receptor excision circle and T-cell dynamics
after allogeneic stem cell transplantation are related to clinical events. Blood. 2002;99:34493453.
16. Kernan NA, Young J, Papadopoulos E, et al. Antithymocyte globulin (ATGAM) and methylprednisolone (MP) promote engraftment of T cell de-
pleted (SBA- E-) BMTs from HLA-identical
siblings. Blood. 1995;86:621a.
17. Kernan NA, Bordignon C, Heller G, et al. Graft
failure after T-cell-depleted human leukocyte antigen identical marrow transplants for leukemia: I.
Analysis of risk factors and results of secondary
transplants. Blood. 1989;74:2227-2236.
18. Bordignon C, Keever CA, Small TN, et al. Graft
failure after T-cell-depleted human leukocyte antigen identical marrow transplants for leukemia: II.
In vitro analyses of host effector mechanisms.
Blood. 1989;74:2237-2243.
19. Collins NH, Bleau SA, Kernan NA, O’Reilly RJ. T
cell depletion of bone marrow by treatment with
soybean agglutinin and sheep red blood cell rosetting. In: Areman HJ, Deeg HJ, Sacher RA,
eds. Bone Marrow and Stem Cell Processing: A
Manual of Current Techniques. Philadelphia, PA:
FA Davis; 1992:171-180.
20. Papadopoulos EB, Carabasi MH, CastroMalaspina H, et al. T-cell-depleted allogeneic
bone marrow transplantation as postremission
therapy for acute myelogenous leukemia: freedom from relapse in the absence of graft-versushost disease. Blood. 1998;91:1083-1090.
21. Przepiorka D, Weisdorf D, Martin P, et al. 1994
Consensus Conference on Acute GVHD Grading.
Bone Marrow Transplant. 1995;15:825-828.
22. Sullivan KM, Agura E, Anasetti C, et al. Chronic
graft-versus-host disease and other late complications of bone marrow transplantation. Semin
Hematol. 1991;28:250-259.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2242
LEWIN et al
23. Cleveland WS, Devlin SJ. Locally weighted regression: an approach to regression analysis by
local fitting. J Am Stat Assoc. 1988;83:596-610.
24. Hastie T, Tibshirani R. Generalized additive models. Stat Sci. 1986;1:297-318.
25. Kernan NA, Collins NH, Juliano L, Cartagena T,
Dupont B, O’Reilly RJ. Clonable T lymphocytes in
T cell-depleted bone marrow transplants correlate
with development of graft-v-host disease. Blood.
1986;68:770-773.
26. Douek DC, Vescio RA, Betts MR, et al. Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of
T-cell reconstitution. Lancet. 2000;355:18751881.
27. Patel DD, Gooding ME, Parrott RE, Curtis KM,
Haynes BF, Buckley RH. Thymic function after
hematopoietic stem-cell transplantation for the
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
treatment of severe combined immunodeficiency.
N Engl J Med. 2000;342:1325-1332.
28. Weinberg K, Blazar BR, Wagner JE, et al. Factors
affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood. 2001;97:
1458-1466.
29. Savage WJ, Bleesing JJ, Douek D, et al. Lymphocyte reconstitution following non-myeloablative
hematopoietic stem cell transplantation follows
two patterns depending on age and donor/recipient chimerism. Bone Marrow Transplant. 2001;
28:463-471.
30. Hochberg EP, Chillemi AC, Wu CJ, et al. Quantitation of T-cell neogenesis in vivo after allogeneic
bone marrow transplantation in adults. Blood.
2001;98:1116-1121.
31. McFarland RD, Douek DC, Koup RA, Picker LJ.
Identification of a human recent thymic emigrant
phenotype. Proc Natl Acad Sci U S A. 2000;97:
4215-4220.
32. De Rosa SC, Herzenberg LA, Roederer M. 11color, 13-parameter flow cytometry: identification
of human naive T cells by phenotype, function,
and T-cell receptor diversity. Nat Med. 2001;7:
245-248.
33. Bell EB, Sparshott SM. Interconversion of CD45R
subsets of CD4 T cells in vivo. Nature. 1990;348:
163-166.
34. Hargreaves M, Bell EB. Identical expression of
CD45R isoforms by CD45RC⫹ ‘revertant’
memory and CD45RC⫹ naive CD4 T cells. Immunology. 1997;91:323-330.
35. Dulude G, Roy DC, Perreault C. The effect of
graft-versus-host disease on T cell production
and homeostasis. J Exp Med. 1999;189:13291342.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2002 100: 2235-2242
Direct evidence for new T-cell generation by patients after either T-cell−
depleted or unmodified allogeneic hematopoietic stem cell
transplantations: Presented in part in abstract form at the 42nd Annual
Meeting of the American Society of Hematology, San Francisco, CA,
December 2000.
Sharon R. Lewin, Glenn Heller, Linqi Zhang, Elaine Rodrigues, Eva Skulsky, Marcel R. M. van den Brink,
Trudy N. Small, Nancy A. Kernan, Richard J. O'Reilly, David D. Ho and James W. Young
Updated information and services can be found at:
http://www.bloodjournal.org/content/100/6/2235.full.html
Articles on similar topics can be found in the following Blood collections
Immunobiology (5489 articles)
Transplantation (2228 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.