Cytomegalovirus Infection in Gambian Infants Leads to Profound CD8
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JOURNAL OF VIROLOGY, June 2007, p. 5766–5776 Vol. 81, No. 11
0022-538X/07/$08.00⫹0 doi:10.1128/JVI.00052-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Cytomegalovirus Infection in Gambian Infants Leads to Profound CD8
T-Cell Differentiation䌤
David J. C. Miles,1* Marianne van der Sande,2 David Jeffries,1 Steve Kaye,3 Jamila Ismaili,4
Olubukola Ojuola,5 Mariama Sanneh,1 Ebrima S. Touray,1 Pauline Waight,6
Sarah Rowland-Jones,1 Hilton Whittle,1 and Arnaud Marchant7
MRC Laboratories Gambia, P.O. Box 273, Banjul, The Gambia1; National Institute for Public Health and the Environment (RIVM),
Bilthoven, The Netherlands2; Jefferiss Trust Laboratory, Imperial College London, London, United Kingdom3;
I.B.L. Institut Pasteur, Lille, France4; Department of Pediatrics, Bronx Lebanon Hospital Center,
1650 Selwyn Avenue, Bronx, New York 104575; Immunisation Department, Health Protection Agency
Centre for Infections, London, United Kingdom6; and Institute for Medical Immunology,
Université Libre de Bruxelles, Charleroi, Belgium7
Downloaded from http://jvi.asm.org/ on May 7, 2021 by guest
Received 9 January 2007/Accepted 9 March 2007
Cytomegalovirus (CMV) infection is endemic in Gambian infants, with 62% infected by 3 months and 85%
by 12 months of age. We studied the CD8 T-cell responses of infants to CMV following primary infection.
CMV-specific CD8 T cells, identified with tetramers, showed a fully differentiated phenotype (CD28ⴚ CD62Lⴚ
CD95ⴙ perforinⴙ granzyme Aⴙ Bcl-2low). Strikingly, the overall CD8 T-cell population developed a similar
phenotype following CMV infection, which persisted for at least 12 months. In contrast, primary infection was
accompanied by up-regulation of markers of activation (CD45R0 and HLA-D) on both CMV-specific cells and
the overall CD8 T-cell population and division (Ki-67) of specific cells, but neither pattern persisted. At 12
months of age, the CD8 T-cell population of CMV-infected infants was more differentiated than that of
uninfected infants. Although the subpopulation of CMV-specific cells remained constant, the CMV peptide-
specific gamma interferon response was lower in younger infants and increased with age. As the CD8 T-cell
phenotype induced by CMV is indicative of immune dysfunction in the elderly, the existence of a similar
phenotype in large numbers of Gambian infants raises the question of whether CMV induces a similarly
deleterious effect.
Human cytomegalovirus (CMV) is endemic throughout the sions of CMV-specific T cells. An average of around 4% of
world, with infection typically occurring earlier in life in low- CD8 T cells can produce gamma interferon (IFN-␥) in re-
income countries than in the industrialized nations where it has sponse to CMV (39). This probably underestimates the size of
been most intensively studied (29). Congenital infection is of- the CMV-specific CD8 population, as clones of a similar mag-
ten associated with pathology, while early postnatal infection, nitude that are specific for single peptides have been detected
which is usually transmitted by breast-feeding, is normally by tetramer staining in children and adults (8, 24). The pro-
asymptomatic (17). found effect that CMV has on the overall CD8 T-cell popula-
The CD8 T-cell response has been associated with protec- tion is evident from the substantially higher proportion of
tion from CMV disease following some organ transplants (5, 6, differentiated CD28⫺ cells present in CMV-infected compared
44). However, a vigorous CD8 T-cell response alone fails to to non-CMV-infected adults (26, 28, 48).
control primary CMV infection transmitted during kidney Considerably fewer studies have been carried out on chil-
transplantation. dren than adults, although the accumulation of differentiated
The phenotype of CMV-specific CD8 T cells in chronic CD28⫺ CD8 T cells has been shown in children as young as 18
infection is well documented and shows characteristics distinct months (8) and in umbilical cord blood samples from congen-
from cells specific for other viruses. The vast majority lose itally infected infants (27). In the latter study, the CD8 T cells
expression of the B7 ligand CD28, and a large number lose showed cytolytic activity and were able to produce IFN-␥,
expression of the costimulatory molecule CD27 (1) and express implying that their mature phenotype was matched by some
the RA isoform of CD45 during chronic infection (7, 50). degree of functionality. However, studies on other viral infec-
Many CMV-specific CD8 T cells express Ki-67 in the early tions have shown that infant CD8 T cells are less able to
stages of infection (10), indicating that they are in the process produce IFN-␥ (19, 25, 35) in response to antigenic stimulation
of mitotic division (12), and the number of dividing cells wanes than those of adults, implying that infant CD8 T cells are not
over time. as functionally mature as their phenotype suggests.
Chronic infection with CMV typically leads to large expan- More than half of infants in The Gambia are infected with
CMV by 6 months of age (2). Consequently, CMV is one of the
first challenges that their immune systems encounter, and in-
* Corresponding author. Present address: MLW Clinical Research
Programme, P.O. Box 30096, Chichiri, Blantyre 3, Malawi. Phone: 265
vestigation of the response of infant T cells to CMV may offer
1 87 6444. Fax: 265 1 87 5774. E-mail: djcm1@liverpool.ac.uk. insights into how infants respond to natural infections.
䌤
Published ahead of print on 21 March 2007. We recruited subjects from a birth cohort established in
5766VOL. 81, 2007 INFANT CD8 T-CELL RESPONSE TO CMV 5767
Sukuta, The Gambia, in order to determine the onset of in- objective criteria exist for the definition of the subpopulations described in the
fection in infants and to evaluate the subsequent CD8 T-cell present study, we made the assumption that the algorithms were valid if they
recognized the same subpopulations as an experienced flow cytometry operator.
response. Lymphocyte populations were identified using a combination of contouring
and morphological operators on the pixels of the front versus side scatter plot.
After gating this population, similar algorithms were used to identify CD8 T-cell
MATERIALS AND METHODS populations and tetramer⫹ cells. This part of the process was tested on a small
Subjects. Subjects were recruited at birth from the maternity ward of Sukuta number of samples stained for CD3 and CD8, and it was established that while
Health Centre as part of an ongoing birth cohort. Informed consent was obtained there was a substantial overlap between the expression of CD8 on CD3⫺ and
from their mothers and documented by signature or thumb print. Recruitment CD3⫹ cells, ⬎90% of the CD8⫹ cells identified were CD3⫹ T cells, and this
was restricted to healthy singleton infants, defined by a birth weight of at least population included an average of 72% of all CD8⫹ CD3⫹ cells (data not
1.95 kg and no congenital abnormalities. The catchment area of Sukuta Health shown).
Centre is periurban and characterized by low income and crowded living condi- Cells stained with CD27 and CD28, CD27 and perforin, and CD27 and gran-
tions. The human immunodeficiency virus statuses of the study subjects were zyme A were divided into three subpopulations, and cells stained with CD95 and
unknown, but adult prevalence in the region was 1 to 4% at the time of the study CD62L were divided into two subpopulations using a fuzzy c-means clustering
(National AIDS Control Program, unpublished data) and so was not thought to algorithm. The extremities of the population region were defined by the furthest
be a significant confounder of this study. outliers.
Mononuclear cells, plasma, and buffy coats were separated from umbilical Cells expressing Ki-67 and cells expressing either high or low levels of Bcl-2
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cord blood and stored. Limited molecular human leukocyte antigen (HLA) were identified using morphological operators to locate a region of highest event
typing was carried out on the buffy coats (4) in order to identify subjects with density. Where this was not possible, a weighted average of the events was used
HLA-A2, -B7, -B8, and -B35, for which known epitope peptides were available. to separate the two regions.
In total, 258 infants were followed up for 12 months to establish the age at For cells stained with HLA-D and CD45R0, an area of highest event density
CMV infection. A subset of 116 infants was selected for studies on the devel- was identified where possible using algorithms similar to those used for Ki-67 and
opment of the CD8 T-cell response to CMV, which was biased toward HLA Bcl2, and the populations were then classified using a cross-hair placed with
types for which peptides were available. horizontal and vertical positions defined by the extreme borders of the densest
The prevalence of CMV in the adult population was established by serological region.
detection of CMV-specific immunoglobulin G (IgG) in a subsample of 194 For cells stained with CD45RA and CD45R0, a breakpoint regression through
mothers, using ELICYTOK-G enzyme-linked immunosorbent assay (Diasorin). the smoothed trend of CD45RA expression was used to identify at which point
All samples were seropositive. only CD45RA was expressed, thus defining a CD45RAbright CD45R0⫺ popula-
The study was approved by the Medical Research Council-Gambian Govern- tion. To further explore the phenotype of the cells as defined by the expression
ment Ethics Committee. of CD45 isoforms, a line of regression was plotted through the population of cells
Diagnosis of CMV infection and blood-sampling schedule. To determine the expressing both CD45RA and CD45R0, and the midpoint was calculated. A
onset of CMV infection, urine samples were collected at birth, fortnightly for “CD45 ratio” was calculated by dividing the distance from the midpoint of the
the first 3 months of life, and monthly until the age of 12 months and tested for regression line to the border with the CD45RAbright CD45R0⫺ subpopulation by
the presence of CMV DNA by PCR. If it was detected, a second sample was the distance from the midpoint to the event with the lowest expression of
taken immediately to confirm infection, and urine sampling was discontinued. CD45RA as defined by fluorescence intensity. A relatively high value for the
Excretion in the urine is usually continuous for at least 6 months following CD45 ratio indicated a high level of expression of CD45R0 and a low level of
infection (D. J. C. Miles and S. Kaye, unpublished data). Congenital infection expression of CD45RA.
was defined by CMV detection in a urine sample collected in the first 2 weeks of A conservative approach was used for clustering definition, and for each
life. algorithm limits were set to define a successful and meaningful cluster/region
Following CMV diagnosis, a blood sample was taken as soon as possible and definition. Of the 1,886 samples scanned, 130 (6.9%) were discarded because
follow-up samples were scheduled for 4 and 12 months after diagnosis. Samples they could not be characterized after an iterative process in which scans were
were collected in heparin and transported to the laboratory within 4 h. If an manually checked to identify obvious algorithm failure.
infant reached 12 months of age without CMV DNA being detected, a blood For each set of markers, appropriate region statistics were defined, and de-
sample was collected at that time. pending on their distribution, they were summarized using means and medians.
Whole blood was used for flow cytometry, and peripheral blood mononuclear The within-subject repeated measurements were accounted for using panel-
cells (PBMCs) were separated on lymphoprep (Axis-Shield PoC AS) columns for averaged models (fitted using the generalized estimating equation function in
use in enzyme-linked immunospot (ELISpot) assays. Stata 8). Where necessary, the region statistics were appropriately transformed
Flow cytometry. All flow cytometry reagents were from Becton Dickinson. The to improve the normality and constant variance assumptions. Due to the small
CD8 population was identified using peridinin chlorophyll-conjugated anti-CD8 number of time points, the most appropriate correlation structure was the ex-
antibody. Preliminary experiments demonstrated that it was possible to identify changeable model, which acknowledges the correlation but assumes it is the
a pure population of CD3⫹ CD8high T cells and that cells binding tetramers same between all time points. The significance of differences over time was
expressed CD8 at the same high level. Further phenotyping for surface markers quantified by a Wald test, and if significant at the 5% level, appropriate contrasts
was carried out using allophycocyanate-conjugated antibodies to CD27, were calculated and P values were adjusted using a step-down Bonferroni
CD45R0, and CD62L and fluorescein isothiocyanate-conjugated antibodies to method.
CD28, CD45RA, CD95, and the class II HLA-D molecules DR, DP, and DQ. Diagnosis of EBV infection. A subset of plasma samples from 12-month-old
Intracellular markers were identified using fluorescein isothiocyanate-conjugated infants was tested for Epstein-Barr virus (EBV) seropositivity. To allow for the
antibodies to Bcl-2, Ki-67, perforin, and granzyme A. All antibodies were titrated persistence of maternal IgG, an infant was regarded as infected if he/she ex-
prior to use to ensure that they were used at their optimal concentrations. Where pressed IgM to the viral capsid antigen (VCA) at 12 months or a higher level of
possible, CMV-specific cells were identified with phycoerythrin-conjugated B7- IgG to the VCA antigen than was present in umbilical cord plasma collected. The
TPRVGGGAM and A2-NLVPMVATV tetramers, synthesized as previously tests were carried out using the ETI-VCA-G and ETI-EBV-M enzyme-linked
described (13, 27). New batches of tetramer were initially used in parallel with immunosorbent assays (Diasorin).
older batches to ensure consistent staining. Red blood cells were lysed using ELISpot. A kit for the detection of IFN-␥ (Mabtech) was used in ELISpot
FACSlyse solution, and lymphocytes were permeabilized using FACSperm II. assays. The peptides used were the A2-restricted MLNIPSINV, NLVPMVATV,
Statistical analysis of flow cytometry data. As the study involved the collection VLGPISGHV, YILEETSVM, and IAGNSAYEYV; the B7-restricted RPHER
of over 1,800 flow cytometry samples over a prolonged period of time, and the NGFTV, TPRVTGGGAM, and CRVLCCYVL; the B35-restricted IPSIN
study design involved both cross-sectional and longitudinal data, objectively VHHY and VFPTKDVAL; and the B8-restricted DANDIYRIF (11, 22, 33, 38,
defining subpopulations of interest was a major challenge. In order to eliminate 42, 47, 49).
the inconsistencies and possible bias inherent in a human operator repetitively A detectable response was defined as a mean treatment spot count that was
analyzing large amounts of data, automatic algorithms were used to define more than 10 spots 105 cells⫺1 greater than twice the mean negative control.
clusters for various combinations of markers using MATLAB 6.1 (The Math- These criteria have previously been found to provide high specificity while al-
works Inc). For each marker set, the same algorithm was applied to each subject lowing reasonable sensitivity in the detection of peptide-specific responses (18).
to maintain objectivity and consistency of the flow cytometry analysis. As no Responses were measured as specific spot-forming units, calculated as the dif-5768 MILES ET AL. J. VIROL.
ference between the mean treatment spot count and the mean negative control the subpopulation that expressed both isoforms was not differ-
spot count. Data were analyzed using Stata 8 and MINITAB 14. ent between tetramer⫹ cells and the overall CD8 T-cell pop-
ulation (Table 1 and Fig. 1C), indicating that while a higher
RESULTS proportion of the tetramer⫹ subpopulation expressed CD45R0
than the overall CD8 T-cell population, the level of CD45R0
Most infants are CMV infected by 12 months of age. Con- expression did not differ between these populations.
genital infection, defined as excretion of CMV in the urine The proportion of cells expressing both isoforms was at its
within the first 2 weeks of life, was detected in 4.1% of the highest immediately postdiagnosis and was substantially lower
infants. Prevalence increased most rapidly between 6 and 12 in the samples taken 4 and 12 months later (P ⫽ 0.012) (Table
weeks of age, by which time 62% were infected. Thereafter, 1 and Fig. 1B). The CD45 ratio showed the same pattern (P ⫽
prevalence continued to rise steadily until sampling concluded 0.008), indicating that not only the proportion of cells that
at 12 months, by which time 85% were infected (unpublished expressed CD45R0 but also the level of CD45R0 expression
data). was higher immediately after diagnosis than in the following
Sampling for CD8 T-cell studies. Longitudinal studies on months.
the CD8 T-cell response to CMV infection were carried out on Cells were also stained with CD45R0 and HLA-D, and the
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98 infants. From these subjects, 64 samples were taken imme- cells that expressed either or both were identified. As with
diately after diagnosis, 92 4 months later, and 79 12 months CD45R0, the proportion of cells that expressed HLA-D was
later, so that 235 of the 294 scheduled samples were collected greater among tetramer⫹ cells than among the whole CD8
successfully. A further 18 samples were collected from infants T-cell population (P ⬍ 0.0001), and expression was higher
who had reached 12 months of age without being infected with immediately after diagnosis than 4 or 12 months later (P ⫽
CMV, so the total cohort numbered 116 infants. 0.004) (Table 1 and Fig. 1D and E). The proportion of cells
Frequencies of peptide-specific CD8 T cells were very vari- expressing both CD45R0 and HLA-D molecules followed the
able. Of 67 subjects who were identified as expressing HLA- same pattern (Table 1 and Fig. 1F).
A2, at least one sample containing CD8 T cells that bound the The proportion of CD8 T cells in the mitotic cycle was
A2-NLVP tetramer was collected from 20 (30%). Of the 37
highest immediately after infection. The age at the time of
subjects who expressed HLA-B7, at least one sample that
diagnosis did not affect the proportion of cells expressing Ki-67
bound the B7-TPRV tetramer was collected from 16 (43%).
(i.e., in the mitotic cycle) (12) at any of the sampling times
The mean frequency of CD8 T cells that bound either tetramer
(data not shown).
was 3.4%, but there was considerable variability, with frequen-
Mitosis was the only variable for which the development of
cies ranging from 0.05% to as high as 32%.
the tetramer⫹ population followed a different pattern than the
The age of CMV infection had no influence on the frequency
overall CD8 T-cell population. Immediately after diagnosis,
of tetramer⫹ cells, and there were no significant differences in
the tetramer⫹ cells exhibited significantly more mitosis than in
frequency between the samples collected immediately after diag-
either of the two subsequent samples (P ⫽ 0.0006) (Table 1
nosis and those collected 4 or 12 months later (data not shown).
and Fig. 2A and B), and more tetramer⫹ cells were undergoing
The phenotypes of tetramerⴙ CD8 T cells followed the same
trends as the overall population. The statistical interaction mitosis than in the overall CD8 T-cell population (P ⬍ 0.0001).
between the phenotypes of tetramer⫹ cells and the overall However, the proportion of tetramer⫹ cells in mitosis halved
population was assessed to establish whether the time elapsed from 31% immediately after diagnosis to 15% 4 months later
since diagnosis had different effects on the phenotypes of tet- and ceased to be different from the overall CD8 T-cell popu-
ramer⫹ cells and the overall population. The only significant lation.
interaction found was in the case of the proportion of cells Among the overall CD8 T-cell population, the highest pro-
expressing Ki-67 (P ⫽ 0.015), which is discussed below. For all portion of CD8 T cells in mitosis never exceeded 12%, and
other parameters measured, changes in the phenotype of the there were no significant differences between the proportion of
tetramer⫹ cells were reflected by changes in the phenotype of cells expressing Ki-67 at different sampling times (Table 1 and
the overall CD8 T-cell population, although the actual propor- Fig. 2B).
tions of cells in different subpopulations at any given sampling Expression of CD62L, CD95, and Bcl-2 remained constant
time were often very different. in the year following infection with CMV. The CD8 T-cell
Age at time of diagnosis had no effect on the magnitudes of population was divided into an undifferentiated CD62L⫹ sub-
either the tetramer⫹ subpopulations or any of the subpopula- population (20, 43), expressing various levels of CD95 (Fas),
tions defined by phenotype (data not shown). and a differentiated CD62L⫺ subpopulation, in which nearly
Activation was highest in the early stages of infection in all cells expressed CD95 (Fig. 3A and B). Nearly all of the
tetramerⴙ cells and the overall CD8 T-cell population. Cells tetramer⫹ cells were in the latter subpopulation (P ⬍ 0.0001).
were stained for CD45RA and CD45R0, and a breakpoint Differentiation status could also be defined by high and low
regression was used to divide them into a CD45RAbright levels of Bcl-2 expression, and nearly all tetramer⫹ cells were
CD45R0⫺ subpopulation and a subpopulation expressing ap- in the more differentiated Bcl-2low subpopulation (P ⬍ 0.0001)
proximately reciprocal proportions of both isoforms. Nearly all (23) (Table 1 and Fig. 3C and D).
tetramer⫹ cells expressed CD45R0 (P ⬍ 0.0001), which is In contrast to CD45 isoforms, HLA-D, and Ki-67, the rela-
typically expressed by functional memory or effector cells (37, tive sizes of the subpopulations defined by CD62L and Bcl-2
43), considerably more than the overall CD8 T-cell population remained constant for 12 months following diagnosis in both
(Table 1 and Fig. 1A and B). However, the CD45 ratio among the tetramer⫹ cells and the overall CD8 T-cell population.VOL. 81, 2007 INFANT CD8 T-CELL RESPONSE TO CMV 5769
TABLE 1. Relative sizes of subpopulations of interest within the CD8 T-cell population
Size
Subpopulation CD8 T-cell population Infected (time postdiagnosis)a Uninfected
Immediately 4 mo 12 mo at 12 mo
CD45RAbright CD45R0⫺ Tetramer⫹ 10.3 ⫾ 8.1Ab 14.5 ⫾ 7.6Bb 18.8 ⫾ 10.5Bb
Overall 33.0 ⫾ 11.1A 37.7 ⫾ 10.9B 40.9 ⫾ 10.8B 41.3 ⫾ 19.3
HLA-D⫹ Tetramer⫹ 67.5 ⫾ 24.4Ab 55.1 ⫾ 26.1Bb 56.6 ⫾ 23.9Bb
Overall 28.3 ⫾ 16.6A 23.6 ⫾ 11.8B 24.0 ⫾ 13.6B 22.4 ⫾ 20.1
CD45RO⫹ HLA-D⫹ Tetramer⫹ 42.0 ⫾ 26.0Ab 30.8 ⫾ 20.1Bb 34.3 ⫾ 20.1Bb
Overall 14.3 ⫾ 11.6A 10.7 ⫾ 7.6B 9.1 ⫾ 6.9B 9.9 ⫾ 11.8
Ki-67⫹ Tetramer⫹ 31.3 ⫾ 22.3Ab 14.5 ⫾ 14.5B 10.4 ⫾ 9.9B
Overall 12.5 ⫾ 9.7 8.4 ⫾ 5.2A 8.5 ⫾ 5.5A 13.4 ⫾ 14.7
CD62L⫹ Tetramer⫹ 16.6 ⫾ 13.0Ab 10.3 ⫾ 8.9Ab 11.9 ⫾ 16.3Ab
Overall 64.8 ⫾ 12.4A 62.6 ⫾ 14.9A 64.8 ⫾ 14.7A 73.2 ⫾ 13.4c
Bcl-2low Tetramer⫹ 97.1 ⫾ 3.2Ab 89.5 ⫾ 15.5Ab 84.3 ⫾ 23.6Ab
Overall 56.0 ⫾ 19.1A 59.3 ⫾ 19.7A 55.4 ⫾ 22.9A 40.2 ⫾ 18.7
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CD27⫹ CD28⫹ Tetramer⫹ 26.7 ⫾ 26.9Ab 15.6 ⫾ 18.6Ab 14.4 ⫾ 13.0Ab
Overall 59.3 ⫾ 19.4A 60.7 ⫾ 17.9A 62.8 ⫾ 19.8A 86.9 ⫾ 20.4c
CD27⫹ CD28⫺ Tetramer⫹ 61.4 ⫾ 23.9Ab 60.8 ⫾ 18.4ABb 58.3 ⫾ 16.4Bb
Overall 28.4 ⫾ 14.4A 22.9 ⫾ 10.7AB 20.0 ⫾ 9.9A 10.6 ⫾ 16.7c
CD27⫺ CD28⫺ Tetramer⫹ 10.8 ⫾ 5.8Ab 22.2 ⫾ 14.6Bb 26.6 ⫾ 14.5Bb
Overall 12.3 ⫾ 8.1A 16.5 ⫾ 10.2B 17.3 ⫾ 12.4B 2.5 ⫾ 3.8c
CD27⫹ perforin⫺ Tetramer⫹ 31.7 ⫾ 28.0Ab 33.4 ⫾ 28.0Ab 29.9 ⫾ 27.1Ab
Overall 63.5 ⫾ 20.0A 60.8 ⫾ 18.5A 65.1 ⫾ 19.7A 83.8 ⫾ 21.3c
CD27⫹ perforin⫹ Tetramer⫹ 53.7 ⫾ 25.6Ab 44.1 ⫾ 20.9Ab 46.4 ⫾ 20.4Ab
Overall 23.8 ⫾ 13.8A 21.5 ⫾ 10.0A 20.0 ⫾ 13.8A 12.2 ⫾ 16.1c
CD27⫺ perforin⫹ Tetramer⫹ 14.1 ⫾ 8.6Ab 21.8 ⫾ 16.0Bb 22.8 ⫾ 14.9Bb
Overall 12.6 ⫾ 8.4A 17.7 ⫾ 10.2B 14.9 ⫾ 10.2B 4.0 ⫾ 5.3c
CD27⫹ granzyme A⫺ Tetramer⫹ 6.7 ⫾ 9.8Ab 6.4 ⫾ 12.8Ab 2.7 ⫾ 2.7Ab
Overall 51.8 ⫾ 20.0A 51.5 ⫾ 18.4A 52.7 ⫾ 20.6A 69.3 ⫾ 22.8c
CD27⫹ granzyme A⫹ Tetramer⫹ 72.7 ⫾ 20.7Ab 66.7 ⫾ 16.8Bb 67.0 ⫾ 15.1Bb
Overall 33.6 ⫾ 15.5A 27.8 ⫾ 11.8B 26.6 ⫾ 11.8B 24.6 ⫾ 18.9c
CD27⫺ granzyme A⫹ Tetramer⫹ 17.6 ⫾ 10.3Ab 26.1 ⫾ 16.5Bb 29.1 ⫾ 15.9Bb
Overall 14.7 ⫾ 8.7A 20.8 ⫾ 10.8B 20.7 ⫾ 13.5B 6.1 ⫾ 5.1c
a
Represented as mean percentages ⫾ standard deviations. Different uppercase letters are placed next to subpopulations that are significantly different between
sample times.
b
Significant differences in the relative size of the relevant subpopulation between the tetramer⫹ and the overall CD8 T-cell populations.
c
Significant differences between the relative sizes of the relevant population between infants uninfected with CMV at 12 months and infected infants of the same
age.
The proportion of differentiated CD8 T cells is constant in defined by CD27 and CD28 expression remained when the
the year following CMV infection, but CD27 is lost progres- stain for CD28 was replaced with stains for perforin (Table 1
sively. Cells stained for the costimulatory molecules CD27 and and Fig. 4B) or granzyme A (data not shown).
CD28 were divided into naı̈ve (CD27⫹ CD28⫹), intermediate Production of IFN-␥ by peptide-specific cells is impaired in
(CD27⫹ CD28⫺), and fully differentiated (CD27⫺ CD28⫺) younger infants. The IFN-␥ production of the PBMCs of sam-
subpopulations (1, 16, 43). Few tetramer⫹ cells expressed any ples that responded to tetramers was assessed by ELISpot
CD28, so they nearly all fell into the last two subpopulations assay, using the peptide incorporated into the tetramer as the
(Table 1 and Fig. 4A). Among the CD8 T-cell population as a stimulant. The proportion of subjects in which a response was
whole, cells in the intermediate and fully differentiated popu- detected was higher in the samples collected 12 months after
lations nearly all expressed perforin (Table 1 and Fig. 4B) and diagnosis (16/19) than in those collected immediately (6/11) or
granzyme A (data not shown). 4 months (2/11) after diagnosis, though the only significant
Age at diagnosis did not affect the relative distribution of difference was between the 4- and 12-month postdiagnosis
cells between the three populations or their subsequent responses (Fisher’s exact probability test; P ⫽ 0.0012). As
changes in phenotype. Similarly, the proportion of cells ex- frequencies of CMV-specific cells remained constant with time
pressing the naı̈ve phenotype remained constant in the 12 since diagnosis, the increased number of IFN-␥-producing
months following diagnosis. However, there was a progressive, PBMCs indicated an increase in the proportion of functional
albeit nonsignificant, reduction of the intermediate population CD8 T cells. Among the samples in which responses were
and a more pronounced increase in the fully differentiated detected, there were no effects of either age at time of sam-
population among both the tetramer⫹ cells and the overall pling or time since diagnosis on the magnitude of the response
CD8 T-cell population (P ⫽ 0.0003), indicating loss of CD27 (data not shown).
expression from the differentiated CD8 T-cell population (Ta- To establish whether the same trend was apparent in a larger
ble 1 and Fig. 4A). data set, the ELISpot response to peptides with the appropri-
The changes that were apparent when the populations were ate HLA restriction was evaluated using PBMCs from 18 of the5770 MILES ET AL. J. VIROL.
A CD8+Tetramer- p=0.0003 p=0.0009
CD8+Tetramer+ B C
p=0.012 p=0.008
CD45RAbrightCD45RO-
74% 26% 89% 11%
100 6.0
CD45ratio
Percentage
80 4.5
60
3.0
CD45RO
40
1.5
20
0 0
CD45RA n= 62 21 74 24 75 24 62 21 74 24 75 24
Population: total tet+ total tet+ total tet+ total tet+ total tet+ total tet+
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p=0.0003 p=0.0003
D E p=0.004 F p=0.0003
CD45RO+HLA-DR+
12% 10% 23% 25% 100 100
80 80
Percentage
Percentage
HLA-D+
60 60
40 40
CD45RO
7% 17% 20 20
n =0 61 20 76 26 75 24 0 61 20 76 26 75 24
HLA-D Population: total tet+ total tet+ total tet+ total tet+ total tet+ total tet+
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FIG. 1. CMV-specific CD8 T cells are more activated than the overall CD8 T-cell population. (A) Expression of CD45RA and CD45R0 by
tetramer⫹ and tetramer⫺ CD8 T cells, showing the line of best fit and the cutoff used to define the CD45RAbright CD45R0⫺ population. (B) Mean
percentage of CD45RAbright CD45R0⫺ CD8 T cells in tetramer⫹ and total populations for each sampling time. (C) Mean CD45 ratios of tetramer⫹
and total CD8 T cells at each sample time. (D) Expression of CD45R0 and HLA-DR on tetramer⫹ and tetramer⫺ CD8 T cells, showing the cutoffs
used to define the CD45R0⫹ and HLA-DR⫹ populations. (E) Mean percentages of HLA-D⫹ and (F) CD45R0⫹ HLA-DR⫹ CD8 T cells among
tetramer⫹ and total populations at each sample time. The percentages in the scatter plots refer to cells within that quadrant. The boxes indicate
medians and interquartile ranges.
50 infants assessed immediately postdiagnosis who had a de- age, and those sampled 12 months postdiagnosis had been
tectable response to at least one peptide, while 17 of 34 (50%) infected at 4 to 10 weeks of age. There was no difference in the
responded at 4 months postdiagnosis and 29 of 65 (44%) re- magnitude of response by Mann-Whitney U test (Fig. 5D),
sponded at 12 months postdiagnosis, indicating no increase in indicating that age at diagnosis was not a determinant of the
the number of detectable responses over time since diagnosis. response in itself.
However, when the magnitudes of the response in those In order to establish whether there was an effect of age at
cases where the response was detectable at all were considered, sampling, the magnitudes of response at 4 and 12 months
the age at diagnosis correlated with the IFN-␥ response at 12 postdiagnosis were compared longitudinally among the subset
months postdiagnosis (Spearman’s r ⫽ 0.46; n ⫽ 29; P ⫽ described above. Nearly all showed a higher magnitude of
0.012), although not in the samples collected immediately and response at 12 months postdiagnosis than at 4 months postdi-
4 months postdiagnosis (Fig. 5A, B, and C). agnosis (Wilcoxon statistic ⫽ 59; n ⫽ 11; P ⫽ 0.023) (Fig. 6E).
As the age at which the 12-month-postdiagnosis sample was As the increase in the magnitude of the response increased
taken depended on the age at diagnosis, it was necessary to with age at sampling but was not related to the age at diagno-
establish whether the correlation was a direct consequence of sis, the results indicate that the function of the CD8 T cells was
the age of infection or simply due to IFN-␥ production increas- low in early infancy and increased with age.
ing as the infants aged. To eliminate the effect of age at sam- There was no effect of time since diagnosis on the magni-
pling on the magnitude of the IFN-␥ response, a subset of tudes.
infants who were all the same age of 40 to 60 weeks was The range of peptides recognized does not alter with age.
identified, but it included infants that had been infected either The number of subjects with the appropriate HLA type that
4 or 12 months previously. The infants who were sampled 4 responded to any given peptide at any given time point varied
months postdiagnosis had been infected at 17 to 39 weeks of from 0 to 59%, with an average of 18.5%. The PBMC samplesVOL. 81, 2007 INFANT CD8 T-CELL RESPONSE TO CMV 5771
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FIG. 2. CMV-specific CD8 T cells divide more than the overall CD8 T-cell population during the early stages of infection. (A) Expression of
Ki-67 by tetramer⫹ and tetramer⫺ CD8 T cells in a representative sample collected immediately postdiagnosis. (B) The expression of Ki-67 for
all subjects where tetramer staining was carried out, with differences in expression of Ki-67 between tetramer⫹ cells indicated. There were no
differences between the overall populations. The boxes indicate medians and interquartile ranges.
collected immediately after diagnosis responded on average to shown). These differences were not significant by the Kruskal-
16% of the peptides with the appropriate restriction, but sam- Wallis test.
ples collected 4 months later responded to only 12%. A Infection with CMV drives differentiation of the CD8 T-cell
broader response was apparent 12 months later, as PBMCs subset over the first year of life. It was not possible to ascertain
responded to an average of 28% of peptides tested (data not whether the changes in the CD8 T-cell subpopulations were a
FIG. 3. CMV-specific CD8 T cells have a more apoptosis-prone phenotype than the overall CD8 T-cell population. (A) Expression of CD62L
and CD95 on tetramer⫹ and tetramer⫺ CD8 T cells, showing the definition of the CD62L⫹ and CD95⫹ CD62L⫹ populations. (B) Mean
percentages of CD62L⫹ CD8 T cells among tetramer⫹ and tetramer⫺ populations at each sample time. (C) Expression of Bcl-2 on tetramer⫹ and
tetramer⫺ CD8 T cells, showing the definition of the Bcl-2high and Bcl-2low populations. The percentages refer to proportions of Bcl-2low cells.
(D) Mean percentages of Bcl-2low CD8 T cells among tetramer⫹ and tetramer⫺ populations at each sample time. Differences between phenotypes
of the total CD8 T-cell population at different sampling times are indicated. The boxes indicate medians and interquartile ranges. The percentages
in the scatter plots refer to adjacent regions.5772 MILES ET AL. J. VIROL.
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FIG. 4. CMV-specific CD8 T cells are more differentiated and show higher cytotoxic potential than the remainder of the CD8 T-cell population.
(A) Scatter plots of representative samples of tetramer⫹ and tetramer⫺ CD8 T cells stained for CD27 and CD28 with the indicated populations
and the percentage of cells within each population, box plots of all samples indicating medians and interquartile ranges, and differences between
the distributions of all populations between sample times indicated. (B) The same data for cells stained for CD27 and perforin. (C) The same data
for cells stained for CD27 and granzyme A. The populations are identified, and the percentages indicate the proportion of cells in each of them.
consequence of CMV infection or of the ontogeny of the CD8 immune challenges than those that remained uninfected. If
T-cell subset by considering only changes within the CD8 T- CMV status is indeed a correlate of exposure, the same is
cell populations of infected infants. Consequently, the overall probably true of EBV. The EBV status of the subset of infants
CD8 T-cell phenotypes of 51- to 60-week-old uninfected in- that were included in the analyses of the effect of CMV on the
fants were compared with those of infants of a similar age who CD8 T-cell phenotype was determined, and 29 out of 46 (63%)
had been diagnosed at least 43 weeks previously. were found to be infected at the time of sampling. All of the
Over 90% of the CD8 T cells of 10 of 16 (63%) uninfected phenotypes that were different between CMV-infected and
infants from whom data on CD27 and CD28 were collected fell uninfected infants were tested to establish whether there were
into the naı̈ve (CD27⫹ CD28⫹) population, which was a higher similar differences according to EBV status, but none were
proportion of naı̈ve cells than was observed in any CMV- found (data not shown).
infected infant (P ⫽ 0.003) (Table 1 and Fig. 6A). Similar
results were found when the stain for CD28 was replaced with DISCUSSION
a stain for perforin or granzyme A (Fig. 6B and C).
The more differentiated phenotype of the CD8 T cells of The interactions between the CD8 T-cell population of
infants infected with CMV was also indicated by the greater adults and CMV are well documented (1, 10, 13, 26, 39).
expression of CD95 and loss of expression of CD62L (P ⫽ Considerably less information is available on the response of
0.024) (Table 1 and Fig. 6D). No differences in the relative the CD8 T-cell population of infants. We studied the develop-
expression of CD45 isoforms, Bcl-2, HLA-D, or Ki-67 were ment of the CD8 T-cell response of infants in a prospective
detected. cohort. A major technical challenge that we encountered was
The possibility remained that the differences between in- the analysis of the very large volume of flow cytometry data
fants infected or uninfected with CMV were not a direct result produced. There were a number of technical sources of uncer-
of CMV infection but were due to exposure to a wider range of tainty over nearly 3 years of data collection, as well as theVOL. 81, 2007 INFANT CD8 T-CELL RESPONSE TO CMV 5773
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FIG. 5. The magnitude of IFN-␥ responses was greater in older
infants. There was no correlation between age at the time of diagnosis
and the magnitude of IFN-␥ response in those cases where a peptide
induced a peptide-specific response (A) immediately after diagnosis or
(B) 4 months later, but (C) there was a positive correlation by 12
months following diagnosis. (D) Samples collected from subjects be-
tween 40 and 60 weeks of age who had been infected 4 months pre-
viously had an IFN-␥ response similar to that of infants who had been
infected 12 months previously. The bars indicate means. (E) Twelve-
month samples were collected from infants diagnosed with CMV at 4
to 10 weeks when they reached 40 to 60 weeks of age, and 4-month
samples were collected from infants diagnosed at 17 to 39 weeks when
they reached the same age. When the 4- and 12-month samples from
subjects who fell into either of these two categories were compared, a
greater IFN-␥ response was elicited from the 12-month sample in
nearly all cases. The absence of a bar indicates that the sample was
collected but no response was detected.
subjectivity inherent in the usual method of fitting regions FIG. 6. The CD8 T-cell population of CMV-infected infants is more
according to the opinion of the operator. To address these activated and differentiated than that of uninfected infants. (A) Expres-
issues, we used a set of algorithms that defined subpopulations sion of CD27 and CD28 in representative samples and all infants sampled,
drawn from the groups of CMV-infected and non-CMV-infected infants
by applying repeatable criteria. When developing the algo- at 12 months of age. (B) Expression of CD27 and perforin in represen-
rithms, we regarded them as valid if they were in agreement tative samples and all infants sampled, drawn from the groups of CMV-
with the opinions of experienced T-cell biologists, who exam- infected and non-CMV-infected infants at 12 months of age. (C) Expres-
ined every plot produced. The algorithms had the advantage of sion of CD27 and granzyme A in representative samples and all infants
sampled, drawn from the groups of CMV-infected and non-CMV-in-
being able to rapidly and consistently apply the same criteria
fected infants at 12 months of age. (D) Expression of CD62L and CD95
for the definition of a given subpopulation to the entire data in representative samples and all infants sampled, drawn from the groups
set, which was particularly important given the longitudinal of CMV-infected and non-CMV-infected infants at 12 months of age. The
nature of the data collection. percentages in the scatter plots refer to cells within the adjacent region.
Gambian infants are typically infected with CMV within the The boxes indicate medians and interquartile ranges.
first year of life, so CMV is one of the first immunological
insults that they encounter. Studies on neonates infected con-
genitally have shown a mature CD8 T-cell response to CMV tetramer⫹ cells. Further, there was no effect of age at diagnosis
(27), and the suggestion that the infant CD8 T-cell response on any phenotypic marker.
develops in a similar way to that of adults is supported by the Cells specific for CMV showed higher levels of HLA-D and
observation that there was no effect of age on the frequency of CD45R0 than the overall CD8 T-cell population, indicating a5774 MILES ET AL. J. VIROL.
relatively high state of activation. Activation markers declined rate as constant as the expression of CD95 and Bcl-2 would
after the first few months of infection, implying a transition suggest.
from the acute to the chronic phase of infection. It is remark- In the light of the stability of the differentiated CD28⫺
able that the pattern of change with time after diagnosis that population, the reduction in the proportion of cells expressing
was seen in the phenotypes of the tetramer⫹ cells was reflected CD45R0 indicated that a substantial proportion of the differ-
in the overall CD8 T-cell population. entiated cells must be CD28⫺ CD45RAbright CD45R0⫺ by 12
A limitation of the tetramers is that they could identify only months after infection. It has been shown that a high propor-
the subpopulation of CMV-specific cells that responded to the tion of CMV-specific CD8 T cells express CD45RA during
peptide they incorporated, so it was impossible to determine chronic infection (7), and it seems that this population ap-
what proportion of the CMV-specific population was repre- peared gradually over the course of the year following infec-
sented in each individual. As CMV is a large virus composed of tion among both the tetramer binding subpopulations and the
over 200 proteins, that proportion probably varied widely be- overall CD8 T-cell population.
tween individuals. Both the proportion of CD8 T cells that In spite of the emergence of a population of differentiated
respond to chronic CMV infection and the range of available cells immediately after infection, the IFN-␥ responses were
epitopes are typically considerable (39, 45, 46, 49). In the impaired in early infancy, even when the presence of a constant
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present study, the proportion of CMV-specific CD8 T cells was population of cells specific for a given peptide was detected by
probably similarly high, as the median proportion of tetramer⫹ tetramer staining. The difference was particularly evident in
cells was 3.4% and several individuals sustained tetramer⫹ the sample collected 12 months following infection, and the
populations of over 10% of the total CD8 T-cell population lack of difference between children infected for different peri-
throughout the year following infection. ods of time but sampled at the same age indicated that the
The same trends were seen in the overall populations as in difference was due to the age of the infants when sampled
the tetramer⫹ populations in all characteristics that were stud- rather than the age at infection in itself.
ied except for division. It is possible that there was a high Although an IFN-␥ response to CMV peptides has been
enough proportion of CMV-specific cells within the overall detected in the umbilical cord blood of congenitally infected
population to dominate the trends shown by that population, infants (27), the ability of the T cells of infants to produce
although it would be impossible to be certain without a method IFN-␥ in response to antigenic stimuli is impaired by the poor
for identifying all CMV-specific CD8 T cells. However, it is interleukin-12 (14) and CD40 (9) expression of infant dendritic
also possible that CMV infection generated a cytokine envi- cells, and indeed, a reduced IFN-␥ response to human immu-
ronment that nonspecifically affected the activity and differen- nodeficiency virus has been shown in infants below 1 year of
tiation of T cells. Such bystander activation has been demon- age (25, 35). In spite of the phenotypic maturity of the CD8⫹
strated in a number of contexts (21, 30, 40). T-cell responses of some infants, it is evident that the cells were
Tetramer⫹ CD8 T cells took on a differentiated phenotype, as functionally impaired.
defined by loss of CD27, CD28, and CD62L (1, 34, 37, 43), and in The expansion of the overall CD28⫺ CD8 T-cell subpopu-
contrast to the transient expression of markers of activation, such lation observed in infants under 12 months of age raises some
as HLA-D, the proportion of differentiated cells remained stable concerns. A subpopulation of a similar size is characteristic of
for the year following infection. This observation supports the Americans of approximately 70 years of age (15) or 40- to
implication of the consistent CMV-specific subpopulations that a 60-year-old Swedes infected with CMV (28). Further, the pres-
population of CMV-specific CD8 T cells is produced very soon ence of expanded CD28⫺ CD45RA⫹ CD8 T cells is indicative
after infection and maintained thereafter. of failure to respond to influenza vaccination in elderly people
The observation that the relative size of the differentiated (36). Our findings indicate that CMV infection and the pres-
CD8 T-cell population was much smaller in uninfected 12- ence of a large population of CD28⫺ CD45RAbright CD8 T
month-old infants than in infected infants suggests that the cells are so commonplace as to be normal in Gambian infants.
accumulation of differentiated CD8 T cells is largely driven by This is probably the case throughout sub-Saharan Africa, as
CMV itself. Studies on adults have shown that while older our observations are consistent with the finding that large
people tend to have more CD28⫺ CD8 T cells than younger proportions of the CD8 T-cell population of young Ethiopian
people, CMV-infected individuals tend to have a much larger children have lost CD27 expression (41). As CMV infection in
population of CD28⫺ CD8 T cells than their uninfected peers our study commonly occurred before the age of 12 weeks and
(26, 28, 48). Our findings show that such a difference begins immediately induced a CD28⫺ CD8 T-cell population, most
with primary CMV infection, even when this occurs very early infants exhibited indicators for impaired immune function (31)
in life. while receiving childhood vaccinations and before their first
Differentiated CD8 T cells, defined by the loss of CD28, exposure to endemic diseases of infancy, such as malaria,
nearly all expressed perforin and granzyme A, indicating that pneumonia, and rotavirus. Whether CMV infection interferes
the differentiated cells have the potential to function as cyto- with vaccine responses is not known, although it has been
toxic effector cells (27). Further, the proportion of cells ex- suggested that there is a finite amount of immunological
pressing CD95 or low levels of Bcl-2 remained constant for the “space” and that the large clonal expansions of T cells that
year following diagnosis. Although both of these markers are CMV induces may occupy so much of it that there is little room
associated with activation-induced cell death (3), these sub- for the expansion of clones to newly encountered antigens (32).
populations remained constant while the proportion of divid- It is also possible that the presence of a large proportion of
ing cells did not, so it is unlikely that apoptosis took place at a differentiated CD8 T cells and vaccine failure are unrelatedVOL. 81, 2007 INFANT CD8 T-CELL RESPONSE TO CMV 5775
consequences of events at a more fundamental level of the 9. De Wit, D., V. Olislagers, S. Goriely, F. Vermeulen, H. Wagner, M. Goldman,
and F. Willems. 2004. Blood plasmacytoid dendritic cell responses to CpG
immune system. oligodeoxynucleotides are impaired in human newborns. Blood 103:1030–
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paired vaccine responses has not been formally demonstrated 10. Gamadia, L., E. Remmerswaal, J. Weel, F. Bemelman, R. van Lier, and I.
Ten Berge. 2003. Primary immune responses to human CMV: a critical role
and studies of infants in the United States (8) and Japan (24) for IFN-␥-producing CD4⫹ T cells in protection against CMV disease.
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Sissons, S. Rowland-Jones, J. Bell, and P. Moss. 2000. Functional hetero-
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ACKNOWLEDGMENTS 19. Jaimes, M., O. Rojas, A. González, I. Cajiao, A. Charpilienne, P. Pothier, E.
Kohli, H. Greenberg, M. Franco, and J. Angel. 2002. Frequencies of virus-
We thank the staff of the Sukuta birth cohort, Omar Badjie, Fatou specific CD4⫹ and CD8⫹ T lymphocytes secreting gamma interferon after
Bah, Saihou Bobb, Janko Camara, Sulayman Colley, Isatou Drammeh, acute natural rotavirus infection in children and adults. J. Virol. 76:4741–
Mam Maram Drammeh, Baboucarr Jobe, Momodou Jobe, Albert 4749.
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differentiation: implications for vaccine development. Nat. Rev. Immunol.
bou, Sarjo Sanneh, and Mamadi Sidibeh. Akram Zaman, Paul Snell,
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and Sarah Crozier helped with data handling. We are extremely grate- 21. Kanai, T., E. Thomas, Y. Yasutomi, and N. Letvin. 1996. IL-15 stimulates the
ful for the support of Sally Savage of Sukuta Health Centre and Abi expansion of AIDS virus-specific CTL. J. Immunol. 157:3681–3687.
Khan of Western Division Health Team. Louis-Marie Yindom and 22. Kern, F., I. P. Surel, N. Faulhaber, C. Frömmel, J. Schneider-Mergener, C.
Assan Jaye carried out HLA typing. Schönemann, P. Reinke, and H.-D. Volk. 1999. Target structures of the
This work was supported by GSK Biologics and MRC. CD8⫹-T-cell response to human cytomegalovirus: the 72-kilodalton major
immediate-early protein revisited. J. Virol. 73:8179–8184.
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