Sequential Anti-Cytomegalovirus Response Monitoring May Allow Prediction of Cytomegalovirus Reactivation after Allogeneic Stem Cell Transplantation
et al. (2012) Sequential Anti-Cytomegalovirus Response Monitoring May Allow Prediction of
Cytomegalovirus Reactivation after Allogeneic Stem Cell Transplantation. PLoS ONE 7(12): e50248. doi:10.1371/journal.pone.0050248
Sequential Anti-Cytomegalovirus Response Monitoring May Allow Prediction of Cytomegalovirus Reactivation after Allogeneic Stem Cell Transplantation
Sylvia Borchers 0
Melanie Bremm 0
Thomas Lehrnbecher 0
Elke Dammann 0
Brigitte Pabst 0
Benno Wo lk 0
Ruth Esser 0
Meral Yildiz 0
Matthias Eder 0
Michael Stadler 0
Peter Bader 0
Hans Martin 0
Andrea Jarisch 0
Gisbert Schneider 0
Thomas Klingebiel 0
Arnold Ganser 0
Eva M. Weissinger 0
Ulrike Koehl 0
Francesco Dieli, University of Palermo, Italy
0 1 Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School , Hannover, Germany, 2 Pediatric Hematology and Oncology , Johann Wolfgang Goethe-University , Frankfurt, Germany , 3 Institute of Human Genetics, Hannover Medical School , Hannover, Germany , 4 Institute of Virology, Hannover Medical School , Hannover, Germany, 5 Internal Medicine II , Johann Wolfgang Goethe-University , Frankfurt, Germany , 6 Institute of Pharmaceutical Science and Biostatistics, ETH Z u rich, Switzerland, 7 Institute of Cellular Therapeutics, IFB-Tx, Hannover Medical School , Hannover , Germany
Background: Reconstitution of cytomegalovirus-specific CD3+CD8+ T cells (CMV-CTLs) after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary to bring cytomegalovirus (CMV) reactivation under control. However, the parameters determining protective CMV-CTL reconstitution remain unclear to date. Design and Methods: In a prospective tri-center study, CMV-CTL reconstitution was analyzed in the peripheral blood from 278 patients during the year following HSCT using 7 commercially available tetrameric HLA-CMV epitope complexes. All patients included could be monitored with at least CMV-specific tetramer. Results: CMV-CTL reconstitution was detected in 198 patients (71%) after allogeneic HSCT. Most importantly, reconstitution with 1 CMV-CTL per ml blood between day +50 and day +75 post-HSCT discriminated between patients with and without CMV reactivation in the R+/D+ patient group, independent of the CMV-epitope recognized. In addition, CMV-CTLs expanded more daramtaically in patients experiencing only one CMV-reactivation than those without or those with multiple CMV reactivations. Monitoring using at least 2 tetramers was possible in 63% (n = 176) of the patients. The combinations of particular HLA molecules influenced the numbers of CMV-CTLs detected. The highest CMV-CTL count obtained for an individual tetramer also changed over time in 11% of these patients (n = 19) resulting in higher levels of HLA-B*0801 (IE-1) recognizing CMV-CTLs in 14 patients. Conclusions: Our results indicate that 1 CMV-CTL per ml blood between day +50 to +75 marks the beginning of an immune response against CMV in the R+/D+ group. Detection of CMV-CTL expansion thereafter indicates successful resolution of the CMV reactivation. Thus, sequential monitoring of CMV-CTL reconstitution can be used to predict patients at risk for recurrent CMV reactivation.
Funding: This work was supported in part by project 23 (DZIF), (EMW and M. Messerle, Institute of Virology, MHH) and by ref. number: 01EO0802 (IFB-Tx), both
funded by the German Ministry of Education and Research. In addition, the work was supported by the GK1172 (DFG), by Hilfe fu r Krebskranke Kinder Frankfurt
e.V., the Alfred and Angelika Gutermuth-Stiftung, the Dieter-Schlag-Stiftung and by the LOEWE Center for Cell and Gene Therapy Frankfurt (Hessisches
Ministerium fu r Wissenschaft und Kunst, ref. number: III L4-518/17.004, 2010). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
" These authors are joint senior authors.
Reactivation of cytomegalovirus (CMV) remains one of the
major complications after allogeneic hematopoietic stem cell
transplantation (HSCT) [1,2,3]. The latent virus is controlled
mainly by CMV-specific T cells (CMV-CTLs) in healthy persons,
but in immune-compromised patients CMV reactivation occurs
frequently due to impaired T cell reconstitution and post-HSCT
immunosuppressive therapy. CMV reactivation, if not controlled,
can lead to severe and multiple manifestations of CMV disease,
such as CMV retinitis, gastroenteritis or pneumonia . CMV
disease is also associated with a high risk for bacterial or fungal
infections  and development of graft-versus-host disease
(GvHD) [6,7]. Major risk factors for CMV reactivation include
recipient CMV-seropositivity (R+), T cell-depletion (TCD) of the
graft [2,8,9] and pre-established acute GvHD (aGvHD) [7,10].
Monitoring CMV viral load by real-time polymerase chain
reaction (PCR) or by pp65 expression in leukocytes is used to
direct pre-emptive antiviral therapy to reduce the risk for CMV
disease. The high sensitivity of PCR-based methods may invoke
treatment of patients who do not need medication at that time,
thus immunohistochemical detection of pp65 is still used in
addition to PCR . Pre-emptive therapy with ganciclovir
(GCV) has significantly reduced incidence and severity of CMV
disease, but has been associated with severe side effects, the
primary ones being myelo- and/or nephrotoxicity .
Multimers, such as the tetrameric HLA epitope complexes
(tetramer), are commonly used to monitor CMV-specific CTL
reconstitution [13,14,15,16,17,18], and can be used as a tool to
analyze the reconstitution process. Although several studies have
investigated CMV-CTL levels as a possible predictor for CMV
reactivation, no clear protective threshold has yet been defined.
While differences between patient cohorts and transplantation
protocols may make the definition of a reliable threshold value for
therapy initiation or withdrawal difficult, the HLA molecules and
tetramer combinations used to detect CMV-CTLs may contribute
to the variation observed to date in meaningful CMV-CTL levels.
Monitoring time-points or time-frames of patients also differed in
these studies, further complicating the interpretation of the results
[14,15,16,17]. To address these questions, we prospectively
monitored the reconstitution of CMV-specific immune responses
in 278 patients using 7 commercially available tetramers [15,19]
representing various CMV epitopes.
We hypothesized that monitoring using tetramers could be a
valuable tool to individualize antiviral therapy, especially for
patients at increased risk for developing multiple CMV
reactivations. We analyzed factors that influence the CMV-CTL level, to
assess the possibility of identifying the minimal number of
CMVCTLs required to provide protection against reactivation and with
the intent of defining optimal monitoring time-point(s) for
CMVCTL reconstitution in HSCT patients.
Design and Methods
Sample collection and analyses were part of an extended
monitoring program conducted in the course of routine sampling
for clinical follow-up. The study was approved by the University
Hospital Ethics Committees (Hannover and Frankfurt, Germany),
and is registered as #2906 with the Ethics Committee at the
Hannover Medical School and as #50/07 with the Ethics
Committee at the University Hospital Frankfurt. Written informed
consent was obtained from all patients or legal guardians.
Patients were transplanted between January 2006 and
December 2010 in the Departments of Pediatric Hematology and
Oncology and Internal Medicine in Frankfurt and the Department
for Hematology, Hemostasis, Oncology and Stem cell
transplantation at Hannover Medical School. This prospective study
included all patients (n = 278) who had at least one HLA molecule
corresponding to a set of 7 commercially available HLA-CMV
tetramers (Coulter; USA). Patients were transplanted for leukemia,
lymphoma, myelodysplastic or benign hematopoietic dysfunction
syndromes or solid tumors in pediatric patients after the first
relapse of the underlying disease. HLA-matched donors were
available for 78% (n = 218/278) of patients, while 22% (n = 60/
278) received grafts from mismatched donors. Bone marrow (BM;
n = 35), cord blood (CB; n = 2), peripheral blood stem cells (PBSC;
n = 220) or T cell-depleted (TCD) PBSC (n = 21) were used as
grafts. T cell depletion was done ex vivo as described previously
[20,21] using good manufacturing practice (GMP), and reduced T
cells in the graft by 13,000-fold compared to unselected PBSC and
800-fold compared to BM. Sixty-eight percent (n = 190/278) of
the patients were CMV-seropositive (R+), and 73% (n = 139/190)
of this group were transplanted from CMV-seropositive donors
(D+), with the remaining 27% (n = 51/190) receiving grafts from
CMV-seronegative donors (D2). Prior to HSCT, 88 recipients
(32%) were CMV-seronegative (R2), of whom 56% (n = 49/88)
received grafts from CMV-seronegative donors, while 44%
(n = 39/88) were transplanted from CMV-seropositive donors.
Patient and graft characteristics are summarized in Table 1.
GvHD prophylaxis included OKT3 (Janssen-Cilag, Netherlands),
thymoglobulin (Genzyme; USA) or antithymocyte globulin (ATG,
Fresenius, Germany) in combination with cyclosporine A (CsA),
mycophenolate mofetil (MMF) or methotrexate (MTX),
Detection of CMV infection/reactivation after allogeneic
Blood/serum samples from all patients were routinely
monitored for CMV DNA load using PCR  or for pp65 expressing
cells per 400,000 leukocytes in peripheral blood mononuclear cells
(PBMNCs) using immunohistochemistry . Pre-emptive
antiviral therapy was initiated when a) the CMV DNA load increased by
more than 0.5 log levels above the baseline, b) more than 2
pp65expressing cells were present per 400,000 leukocytes in 2
consecutive tests or c) more than 5 pp65-expressing cells were
present per 400,000 leukocytes in a single test. The initial therapy
was ganciclovir (GCV; 5 mg/kg twice a day), which was adjusted
according to the presence of pp65-expressing PBMNC or CMV
DNA load .
Flow cytometric quantification of CMV-CTLs
We monitored patients on days +30, +60, +90, +120 and +200
(all time-points +/215 days) after HSCT. Patients who a)
experienced CMV reactivation or b) at increased risk for
reactivation due to increased immunosuppression were monitored
weekly. Weekly monitoring was stopped when a) CMV
reactivation was resolved or b) immunosuppression reduced. CMV-CTLs
were quantified using HLA-CMV epitope tetramers as previously
described [13,18]. HLA-typing of patients and donors was
conducted during preparation for HSCT via high-resolution
multiplexed PCR .
CD3+CD8+ T cells were quantified using 100 ml of
EDTAanticoagulated blood stained with 10 ml CD3-PCy5/7-labelled
anti-CD3 antibody (Beckman Coulter, Germany), 10 ml
FITClabelled anti-CD8 clone T8 antibody (Beckman Coulter,
Germany), 20 ml PE-labelled anti-CD4 antibody (Beckman Coulter,
Germany) and fluorescent beads (FlowCountTM beads, Beckman
Coulter, Germany) added according to the manufacturers
instructions. CMV-CTLs were quantified using the following
commercially available set of PE-labeled tetramers:
HLA-A*0101VTEHDTLLY, pp50 amino acids (aa) 243255;
HLA-A*0201NLVPMVATV, pp65 aa 495503;
HLA-A*1101ATVQGQNLK, pp65 aa 501509;
HLA-A*2402-QYDPVAALF, pp65 aa 341349; HLA-B*0702-TPRVTGGGAM,
pp65 aa 417426; HLA-B*0801-ELRRKMMYM, IE-1 aa 199
207; HLA-B*3501-IPSINVHHY, pp65 aa 123131 (all:
Beckmann-Coulter, Germany). Each tetramer corresponding to a HLA
Matched tetramers per patient
other diseases with indication for HSCT
CMV reactivations/CMV DeNovo Infection in the various
CMV-serostatus recipient (R)/donor (D) group
CD34 *106/kgBW (mean):
CD3 *106/kgBW (mean):
CD34 *106/kgBW (mean):
CD3 *106/kgBW (mean):
CD34 *106/kgBW (mean):
CD3 *106/kgBW (mean):
n = 278
molecule expressed in the patient was measured separately and
required 200 ml EDTA-anticoagulated blood. Aliquots were
stained with 10 ml aCD3, 10 ml aCD8 and 5 ml tetramer. One
negative control was performed per patient using 200 ml of
EDTAanticoagulated blood and 5 ml negative control tetramer, to which
none of the cells should specifically bind to, provided by the
manufacturer (Beckman Coulter, Germany). All samples were
labeled for 30 min at room temperature (RT), followed by
erythrocyte lysis (15 min, RT) and fixation. The gating strategy
and staining results are shown in Figure 1. At least 10,000
lymphocytes, 1000 CD3+CD8+ cells and 10 tetramer-positive cells
were counted for each tetramer-stained sample. At least 10,000
lymphocytes and 1000 CD3+CD8+ cells were counted for each
negative control tetramer-stained sample and in addition 1100
fluorospheres were counted for each CD3+CD8+ T cell
Absolute numbers of CMV-CTLs were calculated using
fluorescent beads (FlowCountTM beads, Beckman Coulter,
Germany) in a single-platform, no-wash analysis according to
the manufacturers directions. Briefly, samples were washed and
analyzed, after erythrocyte lysis (VersaLyse, Lysing Solution; IO 3,
Fixative Solution 106; Beckman Coulter, Germany), on a FC500
flow cytometer (Beckman Coulter, Germany) . The
absolute number of CMV-CTLs in a sample was calculated by
subtracting negative-control-tetramer-binding cells from
CMVCTLs binding only to CMV-tetramers. Table 1 summarizes the
tetramers corresponding to HLA-molecules present in patients.
Quality constraints were determined in our previeous studies
 and we determined that only whole blood samples
containing at least 50 CD3+CD8+ T-cells per ml blood gave
reliable and between centers reproducible results to cytometrically
detect multimer-positive cells at a reliable event rate as detailed
above. The detection limit is 0.05 multimer-positive cells per ml
blood using these quality constraints. Application of these quality
constraints allowed to include 92% (n = 1712) of 1861 samples in
this tri-center trial (Table S1). Low CD3+CD8+ counts below 50/
ml blood in 149 samples (8%) led to the exclusion of generated data
from this analysis.
Data management and statistical analysis
CMV-CTL data were collected and stored in a mySQL
database. The general purpose PHP5 (Personal Home Page tools
5; open source license) scripting language was used for queries in
the database. The mean number of CMV-CTLs from all tests
using one tetramer was calculated for each patient to evaluate
whether monitoring using single tetramers resulted in similar
CMV-CTL counts in patients with detectable immune responses.
For patients monitored with more than 1 tetramer, the
CMVCTL counts obtained for each tetramer were calculated
individually after HSCT. Clinical data were correlated with CMV-CTL
reconstitution. The influence of the presence or absence of
CMVCTL on the occurrence of CMV reactivation and vice versa was
evaluated. CMV-CTL reconstitution was analyzed at different
Figure 1. Gating strategy for detection of tetramer-stained CMV-CTLs. The gating hierarchy and tetramer positive cells in a patient
expressing 3 of the HLA molecules represented in our tetramer set are shown. The lymphocytes were gated in the FS/SS quadrant (upper left)
followed by the selection of CD3+CD8+-positive cells (lower left). Within the CD3+CD8+-positive population, the percentage of cells that bind each
tetramer is determined in individual samples (200 ml whole blood each). Staining results using the negative control tetramer, HLA-A*0101 CMV
tetramer, HLA-B*0702 CMV tetramer and HLA-B*0801 CMV tetramer are shown. The number of CMV-CTLs per ml blood is indicated in each tetramer
thresholds: namely .0, $1, 3, 5, 7 or 10 CMV-CTL per ml
blood). CMV-CTL levels before and after 1st CMV reactivation
were counted, and the resulting slope between this 2 time-points
was calculated. For the determination of CMV-CTL expansion
after CMV reactivation, the following patient/donor pairs were
excluded: a) CMV-seronegative patients who were transplanted
from seronegative donors and did not develop de novo CMV
infections during follow-up; b) patients who died before day +100,
c) patients with early relapse of the underlying disease by day +100
and d) patients for whom sampling could not be achieved prior to
and after CMV reactivation. Statistical and Kaplan-Meier
analyses were performed with GraphPad Prism 4 and 5
(GraphPad Software, San Diego, USA). Graphs were plotted
using GraphPad Prism 4 and 5). P-values#0.05 were considered
significant, and the significance test applied is indicated for all
pvalues in the figure legends.
CMV-CTL levels vary depending on the tetramer used for
detection, on HLA-molecules expressed and on
occurrence of CMV reactivation
CMV-CTL reconstitution was observed in 71% (n = 198/278)
of the patients. The median level of CMV-CTLs varied
considerably for each HLA allele investigated, ranging from 2
30 CMV-CTLs per ml blood (Figure 2A). CMV-CTLs recognizing
the HLA-A*2402 tetramer were detected at significantly lower
levels in all patients with this HLA molecule compared to
CMVCTLs corresponding to other CMV epitope and tetramer
combinations. In addition, 73% of all FACS analyses with the
HLA-A*2402 tetramer detected no CMV-CTLs per ml blood, and
HLA-A*2402-specific CMV-CTLs did not correlate with the total
number of CD3+CD8+ T cells observed. CMV reactivations did
not occur more frequently in patients with HLA-A*2402. In
contrast, CMV-CTL levels detected by HLA-A*0101,
HLAA*0201, HLA-B*0702, HLA-B*0801 and HLA-B*3501 tetramers
correlated with overall CD3+CD8+ immune reconstitution
(0.5,r,0.8; p,0.0001). The HLA-A*1101-pp65 tetramer was
used to monitor only one patient, thus data obtained with this
tetramer are not shown in Figure 2.
CMV-reactivation occurred in 42% (n = 117/278) of the
patients and had a negative impact on overall survival (p,0.04;
Log-rank Test; Figure S1) as expected. After CMV reactivation
the median CMV-CTL levels were always higher independent of
CMV-epitope tetramer combination used (Figure 2B). The
difference in CMV-CTL numbers prior to and after CMV
reactivation was statistically significant for HLA-A*0101 (p,0.001)
and for HLA-A*2402 (p,0.01).
Sixty-three percent of the patients (n = 176/278) could be
monitored with more than one CMV tetramer (Table 1) and
CMV-CTL levels were influenced by the presence of other
HLAtetramer combinations. For example, the level of T cells detected
by HLA-A*0201-pp65 was significantly lower, if the HLA
molecules expressed by the patients corresponded to both
HLAA*0201 and HLA-B*0702 rather than only the HLA-A*0201
molecule (Figure 3A; median: 1.6 versus 18.5 CMV-CTLs per ml
blood; p,0.05; t-Test with Welchs correction). The number of
CMV-CTLs detected by HLA-B*0702 did not differ whether the
patients also expressed the HLA-A*0201 molecule or not (median:
21.6 and 27.8 CMV-CTLs per ml blood; Figure 3A). A change in
the most abundant CMV-CTLs detected with particular tetramers
occurred in 11% (n = 19/176) of the patients monitored with at
least 2 tetramers. We detected 30 alterations of the most abundant
CMV-CTL lines after HSCT. Figure 3B shows a typical example
of such a change. This patient experienced CMV reactivation on
day +41. A shift from HLA-A*0201 to HLA-B*0801 was detected
by day +100, when the level of HLA-B0801 CMV-CTLs rose
above the level of HLA-A0201 CMV-CTL. In 14 patients, 17
changes (57%; 17/30) led to higher levels of HLA-B*0801 (IE-1)
recognizing CMV-CTLs (Table S2) after day +100 post-HSCT.
Interestingly, these increases/decreases of most abundant
CMVCTLs did not correlate with the time of CMV reactivations after
HSCT (Table S2).
Early CMV-CTL reconstitution correlates well with
protection against CMV reactivation in the R+/D+ group
To identify common features for CMV-CTL reconstitution in
our patient cohort, we analyzed the kinetics of CMV-CTL
reconstitution. CMV-seropositive recipients (R+, n = 190/278;
68%) were grouped into patients receiving a transplant from a
CMV-seropositive donor (R+/D+, n = 139/190) or a seronegative
donor (R+/D2, n = 51/190). Monitoring of reconstitution of
CMV-CTLs was initiated on day 30 (+/215 days) post-HSCT in
the R+/D+ group. In the R+D+ group sixty two patients had no
CMV reactivation after HSCT. By day +50 38 patients (62%,
p,0.02) had 1 CMV-CTL/ml blood, by day +75 46 patients (74%,
p,0.04) had achieved 1 CMV-CTL/ml blood. These were
significantly more patients than those reactivating CMV at least
once (37% by day+50; n = 47/77) (Figure 4A). Interestingly,
increasing the threshold level to between 5 and 10 CMV-CTLs
per ml blood did not improve discrimination between patients with
and without CMV reactivations (Figure S2CF). A typical
example for the R+/D+ patient group illustrates early
reconstitution of CMV immunity by day +60 following CMV reactivation
(Figure 4B). Patients from the R+/D+ group without detectable
CMV-CTLs or with detectable CMV-CTLs that did not expand
during or after the first CMV reactivation (12.5% of patients,
n = 20). Those were at risk for multiple CMV reactivations (n = 13)
similar to patients transplanted from seronegative donors. (R+/
D2) showed a delayed reconstitution of CMV immunity, which
occurred on or after day +120 (Figure 4C). There is no significant
difference in number of patients in the R+/D2 group achieving
immune reconstitution with 1 CMV-CTL per ml blood with
(n = 30) or without (n = 21) CMV reactivation. CMV reactivation
was recurrent in all 30 of the 51 patients who did experience
reactivation, and required prolonged antiviral therapy.
Furthermore, low-level preexisting CMV-CTLs did not proliferate upon
CMV reactivation in 27% (n = 14/51) of R+D2 patients. As
expected, reconstitution of CMV-CTLs was delayed in patients who
received TCD grafts, and the course of reconstitution is similar to
R+ patients transplanted from CMV-seronegative donors (Figure
S3). Patients in the groups R2/D+ or R2/D2 were not included
in statistical analyses, since patient numbers were small and CMV
reactivations (R2/D+ 7/39) or de novo infections (R2/D2 3/49)
were rare events (Table 1) and did not contribute to the elucidation
of CMV reactivation in the context of CMV-CTL reconstitution.
To investigate whether incidence, severity and treatment of
aGvHD influenced the numbers of CMV-CTLs detected, we
analyzed the associated data for the R+/D+ group. The number
of CMV-CTL did not differ significantly in patients without or
with aGvHD grade I or II (Figure S4).
Expansion of CMV-CTLs is associated with single but not
multiple CMV reactivations
CMV-CTL levels detected by the different tetramers (Figure 2)
increased after CMV reactivation, thus, defining a time-dependent
threshold of CMV-CTL that protects the patient against CMV
reactivation is difficult. In our patient cohort, the first CMV
reactivation occurred by day +46 (mean; range: +11 to +170) for
patients of the R+/D+ group. Thus, we reasoned that protective
CMV-CTL levels could probably be predicted by monitoring on
days +30 (+/215) and +60 (+/215) (Figure 5). Sixty-five of 139
patients of the R+/D+ group were included in these analyses.
Figure 5A compares the number of CMV-CTLs on days +30 and
+60 in patients who experienced no or only 1 CMV reactivation.
CMV-CTLs hardly expanded in patients without CMV
reactivation. In contrast, CMV-CTL numbers increased significantly
(p,0.004, t-Test with Welchs correction) between days +30
(range: +15 to +45) and +60 (range: +46 to +99) in patients with a
single reactivation of CMV. These data illustrate the impact of the
first CMV reactivation on CMV-CTL expansion and demonstrate
that CMV-CTL levels cannot serve as predictors for the first CMV
reactivation. Thus, we assessed whether the proliferation of
CMVCTLs could act as a predictor for a successful restoration of the
CMV immune response. We calculated the slope of the
CMVCTL expansion between time-points prior to and after
reactivation, and compared CMV-CTL levels in patients without, or with
a single or recurrent CMV reactivations (Figure 5B). The
expansion slope was significantly higher in R+/D+ patients with
only 1 CMV reactivation. In contrast, no significant increase of the
slope was seen between days +30 and +60 in patients experiencing
recurrent CMV reactivation, indicating that CMV-CTL
expansion may be a prerequisite to resolve of CMV reactivation.
CMV reactivation following HSCT is a consequence of the
massive immunosuppression and insufficient lymphocyte
reconstitution, and occurs more frequently after T cell depletion of the
graft or after transplantation of CMV-seropositive patients with
grafts from seronegative donors. CMV reactivations still
contribute significantly to post-HSCT morbidity, despite advancements
made to reduce CMV disease by monitoring for viral reactivation
and pre-emptive therapy.
Monitoring CMV-CTL reconstitution can be achieved in about
85% of all transplanted patients [13,18], using commercially
available CMV-tetramers. A large cohort of 278 patients could be
monitored with the tetramers, 198 (71%) actually developed
detectable CMV-CTLs following HSCT. The CMV-CTL levels
detected varied greatly (11235 per ml blood), and correlated only
weakly with reconstitution of the CD3+CD8+ T cells, as described
previously . Restoration of CMV-specific immunity is
frequently analyzed using tetramer staining [14,15,24]. However,
absolute values for the CMV-CTL levels required to protect the
patient from reactivation are still actively debated to date. Our
data indicate that CMV-CTL numbers vary considerably for
individual combinations of HLA molecules and CMV epitopes.
Many authors focus on the HLA allele that is most common in the
Caucasian population, HLA-A*0201. Using the
HLA-A*0201NLVP tetramer, 10 to 20 CMV-CTLs per ml blood by day +60 were
described as being protective [25,26]. Our data for CMV-CTL
levels detected by HLA-A*0201-NLVP are similar, with a mean
level of 10 CMV-CTL per ml blood (Figure 2). However, analyzing
additional HLA molecules in our large patient cohort showed that
different HLA types yield quite different median values of detectable
CMV-CTLs (Figure 2). We and others observed that CMV-CTL
levels detected by HLA-A*0101 (pp50243255) or HLA-B*3501
(pp65123131) were considerably higher or much lower than
CMVCTL levels detected by HLA-A*2402 (pp65341349), which yielded a
mean of 4 per ml blood (Figure 2) [15,18,19,27]. We detected no
CMV-CTLs using the HLA-A*2402 tetramer in 73% of the tests.
This is in agreement with data published by others, who detected
low levels of CMV-CTLs using the HLA-A*2402 tetramer when
monitoring CMV-CTL reconstitution [19,27,28,29,30,31,32]. We
also identified no correlation between the immune reconstitution of
CD3+CD8+ T cells and CMV-CTL levels detected using the
HLAA*2402 tetramer. Since CMV reactivation did not occur more
frequently in patients expressing the HLA-A*2402 molecule. We
speculate that either lower levels of HLA-A*2402-corresponding
CMV-CTLs are needed provide protection  or the
immuneresponse in HLA-A*2402-positive individuals is dominated by other
epitopes . In addition to the different median levels of
CMVCTLs, we found that expression of different HLA alleles may
interfere with the expansion of T cells specific for other HLA
peptide combinations. For example, in individuals expressing both
alleles HLA-A*0201 and HLA-B*0702, the HLA-B*0702-pp65
CMV-CTL response prevailed . Thus, in these patients
CMVCTLs binding to HLA-B*0702-pp65 may be the dominant response
to CMV-CTLs recognizing HLA-A*0201-NLVP, since the level of
CMV-CTLs binding to HLA-A*0201-NLVP is significantly higher
in patients expressing only the HLA-A*0201 allele (Figure 3). Our
data imply that there may be more interference between the
different HLA molecules and/or CMV epitopes expressed than
monitoring techniques currently in use have detected. We
documented that changes in the most abundant epitope-specific
CMV-CTL population occurred over time. These alterations were
solely dependent on particular HLA molecules. Interestingly, in 14
of 19 patients the response directed against the HLA-B*0801
epitope of the immediate early (IE)-1 protein prevailed. We
hypothesize that a shift from CMV-CTLs recognizing pp65
epitopes to IE epitopes may delineate the shift from short-term to
long-term protection against CMV reactivation [34,35,36,37].
Taken together, the absolute number of CMV-CTLs detected in
patient blood samples appears to be depend on the HLA expressed
in the patient, the tetramers used for detection of CMV-CTLs and
the time-point of the measurement, thus protective CMV-CTL
levels vary considerably. Taken together, a protective CMV-CTL
count cannot be defined, since the CMV-CTL level varies a) for the
different tetramers used in detection, b) for different HLA
combinations expressed in the patient (e.g. A0201 and B0702)
and c) for a single tetramer over time. Thus, more information than
an absolute number of all CMV-CTLs is necessary to define what is
protective against reactivation, and levels defined as protective may
vary considerably based on the tetramer or tetramer set used for
The most important finding of our study was that significantly
more patients who experienced no CMV reactivation had at least
1 CMV-CTL per ml blood before day +75 following HSCT (range:
+50 to +75), compared to patients who experienced CMV
reactivations (Figure 4). Interestingly, increasing the threshold to
510 CMV-CTLs per ml blood did not yield better discrimination
between patients experiencing or not experiencing CMV
reactivation in our cohort. Lillieri et al. and Tormo et al. detected
similarly low thresholds (13 CMV-CTLs per ml blood) using
functional assays for IFN-c and IL-2 secretion or ELISPOT assays
[38,39,40], implying that, indeed, detection of 1 to 3 CMV-CTLs
per ml blood may indicate the hallmark of a functional immunity
In addition to the currently controversial CMV-CTL quantity
that provides protection against viral reactivation, the influence of
CMV reactivations on immune reconstitution is also widely
debated. While Chen et al. argue that CMV reactivation boosts the
reconstitution of CMV-CTLs , others find no influence of
CMV reactivation on CMV-CTL reconstitution . In our large
prospective cohort, patients reactivating CMV (n = 117/278) had
higher median CMV-CTL numbers than patients without CMV
reactivations (n = 161/278), implying a significant influence of
CMV reactivation on levels of CMV-CTLs detected. Since this
expansion of CMV-CTLs after CMV reactivation does not allow
for the definition of a minimal protective CMV-CTL level, even
using only 1 tetramer, we searched for other means to differentiate
between patients in whom CMV reactivation occurred only once,
was recurrent or did not occur at all. We analyzed CMV-CTLs at
two time-points, namely day +30 (+/215 days) and +60 (+/215
days), and measured the expansion of CMV-CTLs within that
time period. Patients with a protective response after the first
CMV reactivation showed a significantly increased expansion of
CMV-CTLs within this time period compared to patients with
recurrent CMV reactivations. Patients without CMV reactivation
also expanded CMV-CTLs during this time interval, but to a
lesser extent than those with 1 CMV reactivation. The inability of
CMV-CTLs to expand after CMV reactivation may be due to
even minor HLA incompatibilities between donor and recipient.
Our results indicate that analyses using a single tetramer at only
one time point, for instance on day +60, do not allow prediction of
pending or, more importantly, recurrent CMV reactivations.
Despite the fact that patients without CMV reactivation showed
an earlier reconstitution of at least 1 CMV-CTL per ml blood, the
wide range of CMV-CTL levels does not allow definition of
definitive protective value that is broadly applicable for all HSCT
patients. However, monitoring the level of CMV-CTL expansion
between days +30 and +60 (+/215 days) after CMV reactivation
can indicate successful restoration of CMV immunity.
In summary, our results show that sequential tetramer
monitoring rather single time point cut offs of the post-transplant
CMV-CTL immune reconstitution allows a more accurate
interpretation of an individual patients response to CMV. In
addition, CMV-CTL expansion after the first CMV reactivation
indicates recurrence of CMV reactivation even in R+/D+ patients
after allogeneic HSCT. Analysis of the CMV-CTL expansion rate
may facilitate implementation of patient-specific antiviral
strategies, including adoptive transfer of CMV-CTLs to recipients
unable to respond to CMV reactivations.
Figure S1 Impact of CMV reactivation on survival. Patients in
whom CMV reactivation occurred (n = 117) had a significantly
lower probability for survival (p,0.04) than patients not
experiencing CMV reactivation (n = 161).
Figure S2 CMV-CTL analysis applying different threshold
levels. (AF) Reconstitution of CMV-CTLs with respect to
reactivation in the R+/D+ patient group using different thresholds
of CMV-CTLs per ml blood. Percentage of patients reaching the
threshold level is plotted against time after HSCT. (AC) Using
thresholds of .0 to $3 CMV-CTLs per ml blood showed
significant differences between patients with and without CMV
reactivation. (DF) Using thresholds of $5 or higher (per ml blood)
detected no significant differences in the reconstitution of
CMVCTLs between patients with and without CMV reactivation. The
asterisk (*) indicates significant differences at day +50; the plus
symbol (+) indicates significant differences at day +75.
Figure S3 Impact of T cell depletion on CMV-CTL
reconstitution. T cell depletion of the graft results in delayed CMV-CTL
reconstitution. CMV-CTL reconstitution in patients receiving T
cell depleted (TCD) grafts (continuous line) compared with
patients received unmodified grafts (dotted line) after HSCT is
plotted against the time in days after HSCT. Significantly fewer
patients receiving a TCD-graft reconstituted CMV-CTLs at early
time-points following HSCT, however, by day +200 the curves
converge. The asterisk (*) indicates significant differences at day
+50; the plus symbol (+) indicates significant differences at day
Figure S4 Influence of aGvHD. Median CMV-CTL levels in
the R+/D+ group were analyzed with regard to the incidence of
aGvHD in the time intervals from day 0 to +49 and days +50 to
+99. The mean of the SCMV-CTLs is shown for each patient in
which CMV-CTL were detected during the interval. (A) Patients
undergoing CMV-reactivation. (B) Patients not experiencing
Table S1 Overview on quality considerations and their
applicability to the current cohort. In order to detect
tetramerpositive cells at a reliable event rate using the FACS analysis the
population requires more than 10 events and the CD8 count must
be above 50 per ml blood (using a 200 ml aliquot for staining).
Complying with these quality constraints guarantees the
theoretical detection of 0.1% tetramer-positive cells (0.05
tetramerpositive cells per ml blood).
Table S2 Switches and clinical characteristics at time of switch.
Overview on the 19 patients with switches: patient number,
tetramers switching (i.e. which tetramer yielded the higher
CMVCTL per ml blood), time of switch, donor type,
CMVreactivation(s), occurrence of GvHD, application of DLI and
relapse are shown. Switches did not correlate with the time clinical
events like e.g. CMV-reactivation or DLI.
Conceived and designed the experiments: SB MB EMW UK. Performed
the experiments: SB MB MY. Analyzed the data: SB MB UK. Contributed
reagents/materials/analysis tools: SB MB TL PB EMW UK. Wrote the
paper: SB MB EMW UK. Performed data base queries: ED BW RE.
Revised the manuscript: TL AJ RE GS. Contributed to statistical analysis:
SB MB BW GS EMW UK. Provided clinical data: ED BP AJ HM MS
ME. Responsible for patient enrollment: AG TK.
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