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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

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903262

mailto:[email protected]

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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

7282 T CELL RESPONSES TO DENGUE VIRUSES

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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 .

The Journal of Immunology 7283

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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).


8/12/2019 J Immunol-2010-Dung-7281-7


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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