j immunol-2010-dung-7281-7
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of October 23, 2013.This information is current as
Children with Dengueto Commencement of Capillary Leakage in
T Cell Responses in Relation+Timing of CD8
Farrar, Bridget Wills and Cameron P. SimmonsTran Tinh Hien, Sarah L. Rowland-Jones, Tao Dong, JeremyThi Van Thuy, Tran Van Ngoc, Nguyen Van Vinh Chau,Nguyen Thi Phuong Dung, Huynh Thi Le Duyen, Nguyen
http://www.jimmunol.org/content/184/12/7281doi: 10.4049/jimmunol.0903262May 2010;
2010; 184:7281-7287; Prepublished online 12 J Immunol
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The Journal of Immunology
Timing of CD8+ T Cell Responses in Relation to
Commencement of Capillary Leakage in Children with Dengue
Nguyen Thi Phuong Dung,* Huynh Thi Le Duyen,* Nguyen Thi Van Thuy,*Tran Van Ngoc,† Nguyen Van Vinh Chau,† Tran Tinh Hien,† Sarah L. Rowland-Jones,‡
Tao Dong,‡ Jeremy Farrar,*,x Bridget Wills,*,x and Cameron P. Simmons*,x
Immune activation is a feature of dengue hemorrhagic fever (DHF) and CD8+ T cell responses in particular have been suggested as
having a role in the vasculopathy that characterizes this disease. By phenotyping CD8+ T cells (CD38+ /HLA-DR+, CD38+ /Ki-67+,
or HLA-DR+ /Ki-67+) in serial blood samples from children with dengue, we found no evidence of increased CD8+ T cell activation
prior to the commencement of resolution of viremia or hemoconcentration. Investigations with MHC class I tetramers to detect
NS3133–142-specific CD8+ T cells in two independent cohorts of children suggested the commencement of hemoconcentration and
thrombocytopenia in DHF patients generally begins before the appearance of measurable frequencies of NS3133–142-specific CD8+
T cells. The temporal mismatch between the appearance of measurable surface activated or NS3133–142-specific CD8+ T cells
suggests that these cells are sequestered at sites of infection, have phenotypes not detected by our approach, or that other
mechanisms independent of CD8
+
T cells are responsible for early triggering of capillary leakage in children with DHF. The Journal of Immunology, 2010, 184: 7281–7287.
Dengue is an acute mosquito-borne disease that is wide-
spread in tropical and subtropical countries. Dengue
hemorrhagic fever (DHF) is a serious form of dengue that
is characterized by a vasculopathy accompanied by altered he-
mostasis. The loss of water and small molecules from the in-
travascular space into surrounding tissues are the physiological
hallmarks of thevasculopathy in DHF and this process beginsin the
first few days of illness (1).
The pathogenesis of dengue is likely multifactoral. Viral traits
as well as host genetic and physiological factors may each
contribute to the clinical phenotype (2–4). In children and adults,epidemiological evidence suggests that two sequential dengue
virus (DENV) infections, with the second infection caused by
a different DENV serotype to the first, is associated with ele-
vated risk (but is not an absolute requirement) for DHF (5–8).
Infants born to dengue immune mothers can also develop DHF as
a result of primary infection (9, 10). The most prominent hy-
pothesis to explain both epidemiological observations is that
non-neutralizing or subneutralizing levels of anti-DENV Abs
(acquired by previous infection or passively in utero) can en-
hance DENV infection in Fc receptor-bearing cells; a hypothesis
that is supported by in vitro (11–14) and some in vivo (animal
model) evidence (11, 15). The mechanisms through which Ab-
dependent enhancement of DENV infection might result in the
characteristic vasculopathy seen in DHF patients is not fully
defined.
Ag-driven immune activation during secondary infection may
be a crucial mechanistic link between high viral burdens and the
vasculopathy of DHF. In particular, the rapid mobilization of
memory serotype cross-reactive memory T cells that release vas-
odilatory inflammatory molecules has been suggested to be im-
portant in secondary infection (16, 17). Consistent with this,
circulating plasma levels of soluble CD8 and T cell-derived mol-
ecules, such as IFN-g and sIL-2R, are higher in DHF patients than
dengue fever (DF) patients at the time of defervescence, albeit not
corrected for the hemoconcentration present in DHF patients (18).
In addition, a higher frequency of surface activated CD8+CD69+
T cells are present in DHF cases during the febrile phase than in
patients with milder disease (19).
Data on DENV-epitope–specific responses during the febrile
phase is sparse. We previously showed that DENV-epitope–
specific responses are difficult to detect in the acute febrile phase
by ELISPOT assay (20). Similarly, CD8+ T cells specific for the
dominant HLA-Ap11–restricted epitope NS3133–142 were present
only at very low frequency during the febrile phase in Thai chil-
dren, but were readily detected in early convalescence (21). Other
studies to characterize DENV-epitope–specific T cell responses
have used PBMCs collected in late convalescence and so it is
difficult to know whether these findings can be extrapolated to the
febrile phase (22, 23).
A systematic description of the kinetics of CD8+ T cell acti-
vation, and particularly DENV-specific CD8+ T cells, in the
context of dynamic changes in viremia and hemoconcentration
would provide further insights into the role of these cells in im-
munity and pathogenesis. To this end, the aim of the current study
was to measure the timing of specific and nonspecific CD8+ T cell
responses and virological and clinically important hematological
events during the febrile phase of dengue.
*Oxford University Clinical Research Unit and †Hospital for Tropical Diseases, HoChi Minh City, Vietnam; ‡Medical Research Council Human Immunology Unit,Weatherall Institute of Molecular Medicine, John Radcliffe Hospital; and xNuffieldDepartment of Clinical Medicine, Centre for Tropical Medicine, University of Ox-ford, Oxford, United Kingdom
Received for publication October 13, 2009. Accepted for publication April 2, 2010.
This work was supported by the Wellcome Trust.
Address correspondence and reprint requests to Dr. Cameron Simmons, OxfordUniversity Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City,Vietnam. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: DENV, dengue virus; DF, dengue fever; DHF,dengue hemorrhagic fever; FD, fever day.
Copyright 2010by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
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Materials and MethodsPatient recruitment
Two prospective studies are described. In the first study, consecutive children(,15 y) with suspected dengue and ,72 h of illness were enrolled ata private clinic in Ho Chi Minh City, Vietnam, between October 2007 andMarch 2008. All patients were examined by the same physician (Dr. TranVan Ngoc). Blood samples were collected daily during the febrile period formeasurement of viremia and routine hematological investigations. T cellphenotyping and tetramer staining was performed every second day. Con-
valescent blood samples were obtained 2–3 wk after first presentation. Theextent of hemoconcentration in dengue cases was determined by comparingthe maximum hematocrit recorded during the acute phase with the baselinehematocrit. Baseline hematocrit was defined as the lowest of either the valuerecorded before day 2 of illness if platelet count was $200,000 (availablefor 6% of patients), or the value recorded at follow-up 2–3 wk after illnessonset (available for 82% of cases), or a mean of age- and sex-matchedhealthy population value (13% of cases).
The second study was a prospective study of children with dengue atPediatric Hospital no.1 andno. 2 between August 2006 andMarch 2007. In-patients .6 mo of age with suspected dengue and fever for ,7 d wereenrolled in the study. All patients were assessed daily by a study physicianand had daily serial hematological measurements and an ultrasound within24 h of defervescence. In study 2, the extent of hemoconcentration wasdetermined by comparing the daily maximum hematocrit recorded duringhospitalization with a mean age- and sex-matched healthy population baseline
hematocrit.The platelet nadir in studies 1 and 2 was defined as the lowest plateletcount recorded in patients with a minimum of four platelet counts. The dayof defervescence was defined as the day the fever dropped below ,37.5˚C(axillary) and remained so for 48 h. For calibration purposes, the day of defervescence was defined as “fever day (FD) 0,” with the day prior todefervescence defined as “FD 21”. The day of illness was as self-reportedby the patient’s parent or guardian. For both studies, if patients receivedlaboratory confirmation of dengue, then World Health Organization clin-ical classification criteria (24) were applied to each case. Written informedconsent was obtained from a parent or guardian of each study participant.The study protocols were approved by the Scientific and Ethical com-mittees at each participating hospital.
Dengue diagnostics
A capture IgM and IgG ELISA assay (Venture Technologies, Sarawak,
Malaysia) that used inactivated viral Ag from DENV1–4 was performed asdescribed previously (25). DENV levels in plasma were measured using aninternally controlled, serotype-specific, real-time RT-PCR assay that hasbeen described previously (26). Results were expressed as cDNA equiv-alents per milliliter of plasma. A diagnosis of confirmed dengue was madeif the PCR was positive and/or if there was evidence of rising IgM titers toDENV Ag in paired plasma specimens and the response to DENV Ag wasgreater than to JEV Ag. The interpretation of primary and secondary se-rological responses was based on the magnitude of IgG ELISA units inearly convalescent plasma samples taking into account the day of illness.The cutoff in IgG ELISA units for distinguishing primary from secondarydengue by day of illness was calibrated using a panel of acute and earlyconvalescent sera from Vietnamese dengue patients that were assayed atthe Centre for Vaccine Development, Mahidol University (Bangkok,Thailand), using a reference IgM and IgG Ag capture ELISA describedpreviously (27).
HLA typing
HLA typing for HLA-Ap11 alleles was performed by PCR-specific se-quence primers as previously described (28). Each PCR reaction containeda unique set of allele-specific PCR primers (amplifying alleles HLA-Ap1101/02/03/04/05/06/07/08/09/11/12/13 alleles), and a set of internalcontrol primers (amplifying a conserved region from the third intron of theHLA-DRB1 locus). The presence of an allele was defined by the presenceof the allele-specific PCR product and negative when only the internalcontrol product is present.
Flow cytometry and tetramers
Flow cytometric analysis of whole-blood stained with fluorochrome-conjugated monoclonal Abs was performed on a FACS Canto II flowcytometer (Becton Dickinson, San Diego, CA). Cell surface staining was
routinely performed on 100 ml fresh whole blood. All Abs were purchasedfrom Becton Dickinson. HLApA-1101 MHC class I tetramers containingthe NS3133–142 epitope from all four DENV serotypes were synthesized as
previously described (21). A comparison of the peptide epitopes used intetramer folding versus the NS3133–142 epitope present in 815 DENV ge-nomes sampled from dengue patients in southern Vietnam between 2001and 2008 indicated there were no mismatches between the selected pep-tides and the NS3133–142 epitope sequences found in DENV-1 (590/590[100%]) or DENV-3 (31/31 [100%]) genome sequences. For DENV-2, therewas only one DENV-2 variant of the NS3133–142 epitope in these Viet-namese sequences that is not represented in our tetramer pool. This variantwas present at a very low frequency (1/194 sequences, 0.5%). For DENV-4,no Vietnamese sequences were available for analysis. The four tetramers
(DV1NS3133–142 GTSGSPIVNR, DV2.1NS3133–142 GTSGSPIIDK, DV2.2NS3133–142 GTSGSPIVDK, and DV3-4NS3133–142 GTSGSPIINR) werepooled at equal concentrations and used to stain fresh whole blood. Briefly,whole blood was stained with pooled PE-labeled NS3133–142 tetramersat 37˚C for 30 min in dark and then APC-conjugated anti-CD8 mAb wasadded for 15 min at 37˚C. Stained blood was then lysed, washed, fixed, andanalyzed by flow cytometry. The limit of detection was set at three timesthe SD of the mean percentage of tetramer positive events in 23 patients withno virological or serological evidence of acute dengue. We verified that thetetramers were capable of binding the relevant TCR by staining a cross-reactive CD8 T cell clone generated from a Vietnamese patient with dengueand described previously (29).
Statistical analysis
The nonparametric Mann-Whitney U test was used to evaluate differencesin continuous data. Two sides p values ,0.05 were considered statistically
significant. The program Prism (version 5.0) was used for analyses(GraphPad Software, San Diego, CA).
ResultsCharacteristics of study population
The characteristics of the 126 patients in whom CD3+CD8+
T cells were phenotyped are described in Table I. These patients
were a subset of 138 consecutively enrolled patients at an out-
patient primary care clinic of whom there were 12 patients who
were not investigated because of inadequate samples (clotted or
insufficient sample). Among the 126 enrolled and investigated
patients, there were 103 patients with laboratory confirmed
DENV infection and 23 patients with other febrile illnesses that
were not dengue (Table I). There were 92 (73%) patients whoreturned for follow-up 15–30 d after enrollment. After applying
World Health Organization criteria (24) to each dengue case,
there were 17 DHF patients and 86 DF patients.
Circulation of surface-activated CD8+ T cells and their
relationship to viremia dynamics
There were 93 patients with a measurable viremia at enrollment,
with DENV-1 the most prevalent serotype detected (49/93 [52%])
(Table I). Staining for intracellular markers of cellular pro-
liferation (nuclear Ag Ki-67) and surface markers of activation
(CD38+ and HLA-DR+) on CD3+CD8+ T cells were performed at
enrollment and every second day on fresh whole blood samples.
Results are shown for DENV-1 and DENV-3 only as the sample
size for other serotypes was small. Double-positive CD3+CD8+
T cells (CD38+HLA-DR+, CD38+Ki-67+, or HLA-DR+Ki-67+)
were increased in blood only when the DENV-1 (Fig. 1 A, 1C , 1E )
or DENV-3 (Fig. 1 B, 1 D, 1F ) viremia had already begun to de-
cline. Indeed, phenotypically activated CD8+ T cells were most
prominent after defervescence, a time when most patients were
aviremic. Collectively, these data suggest the commencement of
clearance of viremia, and a sizable fraction of the resolved vire-
mia, occurs in the absence of measurable peripheral blood T cell
activation as defined by the activation markers used here.
CD8+
T cell activation and DENV-specific CD8+
T cell responses
and their relationship to vascular leakage
Microvascular leakage is a signature feature of DHF. In DHFpatients (n = 17), hemoconcentration began during the febrile
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phase and the median day at which it peaked was 1 d (FD 21)
before defervescence (Fig. 2 A–C ). In contrast, an increased (de-
fined as twice the mean convalescent value) in the percentage of surface activated CD3+CD8+ T cells (CD38+HLA-DR+, CD38+Ki-
67+, or HLA-DR+Ki-67+) was not measurable in DHF patients
until a median FD of 1, (range, 24 to 3), 0.5, (range, 24 to 3), and
1 (range, 24 to 3), respectively (Fig. 2 A–C ), and this was statis-
tically significant for all three T cell populations ( p , 0.001). This
suggested hemoconcentration in DHF patients commenced in theabsence of any increase in the percentage of surface activated
CD8+ T cells in peripheral blood. We deliberately did not stratify
patients further into their primary or secondary serological status
Table I. Characteristics of the patient population investigated for CD8+ T cell phenotype and frequency of NS3133–142-specific T cells
VariableDF (n = 86) Median(Range) or No. (%)
DHF (n = 17) Median(Range) or No. (%)
Other Febrile Illness (n = 23)Median (Range) or No. (%) p Valuea
Male sex, no. (%) 40 (46.5) 12 (70.6) 14 (60.9)Age (y) 11 (4–15) 13 (8–15) 11 (5–14)Day of illnessb 2 (1–4) 3 (1–4)FDb,c
23 (25 to 21) 23 (26 to 21)Infecting serotype, no. (%)
DENV-1 39 (51.3) 10 (58.8)DENV-2 10 (13.2) 3 (17.6)DENV-3 26 (34.2) 4 (5.3)DENV-4 1 (1.3)
Secondary infection, no. (%) 59 (68) 14 (82)Log10 of peak viremia, mean (range),
cDNA copies/ml7.37 (4.45–9.90) 7.81 (5.88–9.63)
Platelet nadir, cells/ ml 116,000 (35,200–270,000) 47,100 (11,600–88,300) ,0.001Maximum hemoconcentration (%) 8 (213 to 54) 27 (22–53) ,0.001
aMann-Whitney U test.bAt time of study enrollment.cThe day of defervescence (axillary temperature) was regarded as FD 0.
FIGURE 1. Relationship between kinetics of viremia and the appearance of surface activated CD8+ T cells in peripheral blood. Shown in each panel are
the mean (695% CI) of DENV-1 (n = 49; A, C , E ) or DENV-3 (n = 30; B, D, F ) viremia levels in serial plasma samples from children by FD. The day of
defervescence was defined as FD 0. In each panel, the box and whisker plots represent the percentage of CD8 + T cells that were double positive for CD38/
HLA-DR ( A, B), CD38/Ki-67 (C , D), or HLA-DR/Ki-67 (E , F ). The number of patients that were evaluated on each day is shown below E and F .
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for this analysis as the majority of DHF patients had secondary
dengue (82%) and our primary end point of interest was capillary
leakage.
To examine DENV-specific responses we used a pool of HLA-class
I tetramers carrying common variants of the HLA-Ap11–restricted
NS3133–142 peptide from DENV-1, DENV-2, and DENV-3/4. The
NS3133–142 epitope is typical of most CD8+ T cell epitopes in DENV
in that across the four serotypes there are several variant sequences.
Also typical is the presence of two or more variant sequences within
a serotype. Supplemental Table I summarizes sequence diversity in
known CD8+ T cell epitopes between and within serotypes in a
global collection of 2148 DENV genome sequences sampled from 31
countries. To detect HLA-Ap11–restricted NS3133–142 T cells, we
stained whole blood samples (median, 4; range, 2–6 samples in-vestigated per patient) from each study participant at enrollment and
then every second day with a pool of tetramers. Of the 103 dengue
patients, 33 patients (32%) had at least one sample with a measurable
NS3133–142-specific tetramer staining population of CD8+ T cells,
including nine dengue patients with DHF. The median FD on which
tetramer positive cells were first detected in the nine DHF patients
was +1 (range, 23 to 12) and this was 2 d later than the mean peak in
hemoconcentration described for these patients in Fig. 2. Among the
33 patients who were ever tetramer positive, NS3133–142-specific
T cells were detected during the febrile phase in less than half (13/33[39%]) the cases. In the early convalescent afebrile phase, measur-
able NS3133–142-specific CD8+ T cells were present in a significantly
greater proportion of patients who were ever positive (23/33 [70%])
( p = 0.02). The percentage of circulating NS3133–142-specific CD8+
T cells during the early convalescence phase was significantly higher
than during the febrile phase (paired t test, p = 0.037) (Fig. 3). These
results suggest NS3133–142-specific CD8+ T cells reach measurable
frequencies around the time of defervescence and after the com-
mencement of hemoconcentration in DHF patients.
Timing of DENV-specific CD8+ T cell responses in relationship
to hematological markers of disease
To explore more fully the relationship between the appearance of
measurable NS3133–142-specific T cells in blood and hemoconcen-tration and thrombocytopenia in a larger patient population, pooled
tetramers were used to stain paired (study enrollment and early
convalescent) peripheral blood samples from 422 children hospi-
talized with suspected dengue, of whom 390 were laboratory con-
firmed. The characteristics of this patient population are described in
Table II. At enrollment, the median illness day was 4 d (range 1–6)
and the median number of days prior to defervescence 2 d (range,
26 to 0). Among the 390 patients with laboratory confirmed den-
gue, there were 62 (16%) patients (41 with DHF and 21 with DF)
with blood samples that were ever positive with the pooled NS3133–
142-specific tetramers. All 62 patients were confirmed as being HLA-
Ap11–positive by PCR-specific sequence primers and 82% of the
DHF patients had secondary dengue (Table II).Daily hematocrit measurements indicated hemoconcentration
(.20%) was first present a median of 1 d (FD range, 25 to 2 d)
FIGURE 2. Relationship between hemoconcentration and the appearance
of surface activated CD3+CD8+ T cells in peripheral blood. Shown in each
panel are the mean (695% CI) percentage levels of hemoconcentration in
serial plasma samples from children with DF (n = 86) or DHF (n = 17) by
FD. Hemoconcentration was measured against autologous convalescent
samples for 82% of patients. In each panel, the bar graph represents the
median (interquartile range) percentage of CD8+ T cells that were double
positive for HLA-DR/Ki-67 ( A), CD38/Ki-67 ( B), or CD38/HLA-DR (C ),
The number of patients that were evaluated on each day for either T cell
phenotype or level of hemoconcentration is shown below C .
FIGURE 3. Frequencies of NS3133–142-specific CD8+ T cells during the
febrile and afebrile phase. Serial whole blood samples from 103 children
with dengue were stained with a pool of tetramers (DENV-1, DENV-2, and
DENV-3/4) specific for NS3133–142-specific CD8+ T cells. The scatterplot
shows the frequency of tetramer staining NS3133–142-specific CD8+ T cells
in individual patient samples according to whether they were detected
during the febrile phase (fever day, 26 to 21), afebrile phase (fever day
0–3), or convalescence (fever day 10–15). The frequency of circulating
DENV-specific CD8+ T cells at early convalescence phase were signifi-
cantly higher than at febrile phase ( p = 0.037, by Mann-Whitney U test).
The lower limit of detection (solid horizontal line) was defined as the mean
plus 3 SD of the frequency of tetramer-positive staining events in blood
samples from patients with no evidence of dengue (62 measurements in
23 nondengue patients).
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prior to defervescence in the 41 patients with DHF that were ever
positive with the pooled NS3133–142-specific tetramers (Fig. 4 A).Thrombocytopenia of ,100,000 cells/mm3 was also first detected
a median of 1 d (FD range: 25 to 2 d) prior to defervescence in the
same patients (Fig. 4 B). It is probable that hemoconcentration
(.20%) and thrombocytopenia (,100,000 cells/mm3) occurred
prior to study enrollment in some patients, leading to an over-
estimate of the time required for these hematological changes to
first occur. In contrast, NS3133–142-specific CD8+ T cells first be-
came detectable in DHF patients on FD +1 (FD range, 23 to 4 d)(Fig. 5 A), a median of 2 d (FD range, 0 to 5 d) after thrombocy-
topenia (,100,000 cells/mm3) was first detected (comparison of
median fever day, p = 0.0001 (Fig. 5 B) and a median of 2 d (FD
range, 0 to 5 d) after hemoconcentration ($20%) was first detected
in each patient (comparison of median FD, p = 0.0001) (Fig. 5C ).
The timing of these events is summarized in Table III. Collectively,
these data indicate NS3133–142-specific CD8+ T cells generally be-
come measurable in the peripheral blood only after the com-
mencement of vascular leakage or thrombocytopenia in patients
with DHF.
Discussion
Secondary heterotypic DENV infections are a risk factor for DHFin children and adults. Anemestic, cross-reactive T cells responses
are a part of the host response to secondary infection and have been
suggested to contribute to immunopathogenesis. The major find-
ings in the current study are that surface activated CD8+ T cells and
NS3133–142-specific CD8+ T cells are generally not measurable in
peripheral blood prior to the commencement of hemoconcentra-
tion, thrombocytopenia, or resolution of viremia and therefore it
seems unlikely they are critical triggers of these events.
Significant immune activation undoubtedly occurs in patients
with DHF and secondary infections. Relatively higher levels of
various proinflammatory cytokines and their receptors and soluble
CD4/8 are found in acute sera of children with DHF compared with
children with DF (18, 30, 31). DHF patients also have a greaterpercentage of CD8+ T cells bearing the early activation marker
CD69+ than DF patients during the febrile phase (19). In the
context of virus-specific responses, Mongkolsapaya et al. detected
very low frequencies of NS3133–142-specific T cells in the late
febrile phase with a subsequent peak in frequency a few weeks
after illness onset (21). The very low-frequency of NS3133–142-
specific T cells during the febrile phase was suggested to reflect
huge proliferation balanced by massive apoptosis (21). However,
these findings were not analyzed in relation to the commencement
of capillary leakage. Our own previous studies have indicated
DENV-epitope–specific T cells are difficult to detect during the
febrile phase by ELISPOT assay, but that responses are readily
measured in early convalescence (20). Other studies of virus-
specific responses demonstrated ELISPOT frequencies of T cellsspecific for the HLA-Bp7–restricted epitope NS3222–230 were
Table II. Characteristic of the patient population in whom tetramer staining was performed for NS3133–142-specific T cells
Tetramer Positive Tetramer Negative
VariableDF (n = 21) Median
(Range)/No. (%)DHF (n = 41) Median
(Range)/No. (%)Dengue (n = 328) Median
(Range)/No. (%)
Male sex, no. (%) 13 (61.9) 25 (60.9) 176Age (y) 10 (3–14) 10 (2–15) 10 (1–15)Day of illness at enrollment 3 (1–4) 3 (1–6) 4 (2–7)FD at enrollmenta 22 (23 to 0) 22 (25 to 0) 22 (26 to 0)Infecting serotype, no. (%)
DENV-1 4 (22.2) 12 (31.6) 186 (63.9)DENV-2 10 (55.6) 19 (50) 83 (28.5)DENV-3 4 (22.2) 7 (18.4) 21 (7.2)DENV-4 0 0 1 (0.3)
Secondary infection, no. (%) 18 (85.7) 34 (82.9) 247Log10 of viremia, mean (range), cDNA copies/mlb 6.82 (3.98–9.77) 7.49 (3.78–12.21) 6.80 (3.03–11.83)Platelet nadir, cells/ ml 61,000 (33,000–124,000) 36,000 (10,000–96,000) 50,000 (1,000–270,000)Maximum hemoconcentration (%) 13.9 (2.2–19.4) 28 (10.5–54.1)c 22 (222.6 to 52.78)
Data are median (range) values, unless otherwise indicated.aThe day of defervescence was regarded as fever day 0.bDENV viremia measured at enrollment.cOne patient without hemoconcentration but with pleural effusion by ultrasound finding.
FIGURE 4. Timing of hemoconcentration in children with DHF. The
box and whisker plots represent percentage hemoconcentration ( A) and
platelet counts ( B) in children with DHF (n = 41) by fever day Levels of
hemoconcentration were determined by comparison of daily hematocrit
values in patients against healthy population age and sex stratified mean
hematocrit values (see Table I). Hemoconcentration of .20% was first
detected a median of 1 d before defervesence in these children. The
number of children with hematocrit or platelet measurements on each fever
day is shown beneath the x-axis.
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higher in the late convalescent phase in patients who had DHF
compared with DF; assessments during the febrile phase were not
performed (22). Collectively, a limitation of previous studies of
T cell responses in dengue is that the temporal relationship be-
tween measurable T cell immune activation and the commence-
ment of vascular leakage (as opposed to when vascular leakage is
at its most severe) has not been directly addressed. This is im-
portant for understanding the triggers for capillary leakage.
In two populations of children with DHF we found that
hemoconcentration and thrombocytopenia first occurred prior to
the appearance in peripheral blood of CD8+ T cells bearing
markers of activation (CD38, HLA-DR) or proliferation (Ki-67).Frequencies of proliferating CD8+ T cells (Ki-67+) were high in
early convalescence and this was consistent with previous studies
that have used this marker (21). NS3133–142-specific CD8+ T cells
were also rarely detected during the febrile phase, and signifi-
cantly, were generally not measurable prior to the detection of
hemoconcentration (.20%) or thrombocytopenia in children with
DHF. The temporal mismatch between the first detection of he-
moconcentration and measurable CD8+ T cells or NS3133–142-
specific CD8+ T cells might suggest these cells have a negligible
role in triggering capillary leakage. An alternative explanation for
the absence or very low frequency of NS3133–142-specific T cellsduring the febrile phase [this study and (21)], is that they are
sequestered in sites of infection or have downregulated TCRs (29).
This would imply a massive expansion and distribution of central
and/or effector memory NS3133–142-specific CD8+ T cells after
a few days of infection and negligible ongoing circulation of
CD8+ T cells expressing the relevant, specific TCR. A second
alternative explanation is that they are sequestered and pro-
liferating in lymphoid tissue during the febrile phase but have yet
to enter the peripheral circulation. Studies to address these issues,
in either primate animal models or more directly in patients (e.g.,
fine biopsy collections), will be needed to resolve these questions.
A plausible role for T cells in the immunopathogensis of sec-
ondary dengue is that they act to amplify an already establishedproinflammatory cascade mediated by innate responses to a large
viral burden. In this two-stage model, a large viral Ag mass
(plausibly mediated by Ab-dependent enhancement) might be
necessary and sufficient to trigger capillary leakage through innate
mechanisms, such as complement activation, cytokine secretion by
activated macrophages/dendritic cells, and/or NS1-mediated per-
tubations of the vascular endothelium. Exacerbation of capillary
leakage that has already commenced might occur when effector
T cells accumulate at sites of infection. Such a model could also
explain DHF in infants with primary infections and no DENV-
specific memory T cells; the innate response (complement acti-
vation, cytokine secretion) may be necessary and sufficient to
trigger clinically significant capillary leakage in infants because of their intrinsically poor capacity to compensate for microvascular
leakage compared with older children or adults (32). Consistent
with this, many of the cytokines found to be elevated in children
with DHF and secondary infection are also elevated in infants with
primary infection (33, 34).
There are several limitations to our study. First, our inves-
tigations of surface markers on CD8+ T cells was not exhaustive
but focused on two well-described markers of activation (HLA-
DR and CD38) and one intracellular marker of cellular pro-
liferation (Ki-67). It is possible that T cells responding to altered
peptide ligands in vivo may not express these surface markers or
enter cell cycle and this should be a focus for future research.
Secondly, we investigated CD8+ T cells only when T regulatory
and conventional CD4+ T cells could also be important (35–37).Third, our analysis of DENV-specific CD8+ T cells was limited to
FIGURE 5. Temporal relationship between detection and frequency of
HLA-Ap1101–restricted NS3133–142-specific CD8+ T cell responses and
hemoconcentration, thrombocytopenia and defervescence in children with
dengue. Shown in each scatterplot is the percentage of NS3133–142-specific
CD8+ T cell detected by tetramer staining in individual patient blood
samples against a reference timepoint of day of defervescence (day 0 in A),
day when platelet count of ,100,000 cells/ ml was first detected in each
patient (day 0 in B), and day when hemoconcentration of .20% was first
detected in each patient (day 0 in C ). The values below the x-axis are thenumber of patients evaluated on each day.
Table III. Timing of hematological events in relation to appearance of
NS3133–142-specific T cells
Event in DHF Patients (n = 41) Median Fever Day (Range) p Value
First hemoconconcentration(.20%)
21 (25 to 2)
First thrombocytopenia(,100,000 cells/mm3)
21 (25 to 2)
First detection of
NS3133–142-specific T cells
+1 (23 to 4) ,0.0001a
aCompared with first hemoconcentration (.20%) and compared with first throm-bocytopenia (,100,000 cells/mm3).
7286 T CELL RESPONSES TO DENGUE VIRUSES
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one dominant epitope (NS3133–142) restricted through the most
common class I HLA alle in the Vietnamese population, HLA-
Ap11. Reponses to other DENV CD8+ T epitopes might occur
with greater rapidity than that described in this study. Finally, our
study was biased toward patients with DENV-1 as this was the
most prevalent serotype in circulation at the time. Nevertheless,
this study provides the first description of a temporal mismatch
between the CD8+ T cell response to a well-characterized epitope
and commencement of capillary leakage. Future studies of T cellresponses in dengue need to consider the timing of responses
relative to the commencement of capillary leakage, generally the
most important clinical complication of dengue.
DisclosusresThe authors have no financial conflicts of interest.
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