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Heterologous Viral Serotypes
Their Response to Variant Epitopes of
Quantitative and Qualitative Differences in
Dengue Virus-Reactive CD8+ T Cells Display
Hema S. Bashyam, Sharone Green and Alan L. Rothman
2006; 176:2817-2824; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Copyright © 2006 by The American Association of
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The Journal of Immunology
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Dengue Virus-Reactive CD8?T Cells Display Quantitative and
Qualitative Differences in Their Response to Variant Epitopes
of Heterologous Viral Serotypes1
Hema S. Bashyam, Sharone Green, and Alan L. Rothman2
Reactivation of serotype cross-reactive CD8?memory T lymphocytes is thought to contribute to the immunopathogenesis of
dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel
HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8?T cells in four donors after primary
vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence
differences in heterologous dengue serotypes on dengue-reactive CD8?memory T cell cytokine and proliferative responses. The
D3 variant of a different NS4b epitope 2423 and the D2 variant of the NS4a epitope 2148 induced the largest cytokine response,
compared with their respective heterologous sequences in all donors regardless of the primary vaccination serotype. Stimulation
with variant peptides also altered the relative frequencies of the various subsets of cells that expressed IFN-?, TNF-?, MIP-1?,
and combinations of these cytokines. These results indicate that the prior infection history of the individual as well as the serotypes
of the primary and heterologous secondary viruses influence the nature of the secondary response. These differences in the effector
functions of serotype cross-reactive memory T cells induced by heterologous variant epitopes, which are both quantitative and
qualitative, may contribute to the clinical outcome of secondary dengue infection. The Journal of Immunology, 2006, 176: 2817–
activation of the cross-reactive T cell component of the memory
pool by unrelated viruses resulted in altered T cell epitope hierar-
chies (1) and reshaped T cell repertoires (2). These changes influ-
enced the ultimate outcome of sequential infection by improving
protective immunity (3) or, in some instances, by enhancing im-
munopathology (4). The mechanisms of heterologous immunity
that may lead to a negative outcome have been inadequately stud-
ied in humans.
Dengue virus infections offer a clear paradigm for investigating
the role of cross-reactive memory T cells during an immune re-
sponse to sequential infections with heterologous viruses. The four
natural variants (serotypes) of dengue virus (1–4) can cause an
acute infection in humans that presents clinically either as classical
dengue fever (DF)3or as the more severe dengue hemorrhagic
fever (DHF) (5, 6). Primary infection with any of the four sero-
types results in lifelong immunity to the same serotype but leaves
the individual susceptible to sequential secondary infections by
everal recent studies have highlighted the importance of T
cell cross-reactivity in mediating immune responses dur-
ing infections with heterologous Ags. In these studies, re-
heterologous serotypes (7). Epidemiological studies in Cuba and
Southeast Asia have shown a strong association between second-
ary dengue infection and DHF (8, 9). These data indicate that
previous immunity to dengue may be a risk factor in the develop-
ment of severe dengue disease (10, 11). The mechanisms proposed
for this effect involve serotype cross-reactive, non-neutralizing
Abs, which can enhance infection, and serotype cross-reactive
memory T lymphocytes.
We and others have postulated that serotype cross-reactive T
cells in the memory repertoire are preferentially activated and ex-
panded during secondary infection and contribute to DHF immu-
nopathogenesis through an enhanced output of effector function
(12, 13). In support of this hypothesis, CD8?T cells from patients
with secondary dengue infection show higher binding to tetramers
with heterologous epitope sequences than to tetramers with se-
quences of the secondary serotype, revealing the higher affinity of
these cells for other presumably previously encountered dengue
serotypes (13). Plasma levels of IFN-?, TNF-?, soluble TNFRs
(sTNFRs), sIL-2R, and sCD8 have been found to be higher in
patients with DHF than in those individuals with DF (14–16).
PBMC from patients with DHF have a higher percentage of
CD69?CD8?cells as well as a higher frequency of dengue
epitope-specific CD8?lymphocytes, compared with PBMC from
patients with DF (17, 18).
In in vitro experiments, heterologous restimulation of dengue-
immune PBMC induced only a partial activation of a subset of
serotype cross-reactive cells specific for a dengue epitope. This
epitope was characterized as a partial agonist in its heterologous
form because it induced poor proliferation and IFN-? production
in a CTL clone while able to sensitize target cells for lysis (19).
Consequently, we have hypothesized that the cross-reactive
epitopes of the secondary heterologous serotypes may act as al-
tered peptide ligands for dengue-specific cross-reactive CD8?T
cells and effect changes in the overall functional response of this
Center for Infectious Disease and Vaccine Research, University of Massachusetts
Medical School, Worcester, MA 01655
Received for publication March 31, 2005. Accepted for publication December
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants U19 AI57319 and
K08 AI01729 (to S. G).
2Address correspondence and reprint requests to Dr. Alan L. Rothman, Center for
Infectious Disease and Vaccine Research, University of Massachusetts Medical
School, Room S5-326, 55 Lake Avenue North, Worcester, MA 01655. E-mail ad-
3Abbreviations used in this paper: DF, dengue fever; DHF, dengue hemorrhagic
fever; s, soluble.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
by guest on June 13, 2013
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2824 ALTERED FUNCTIONAL RESPONSE TO DENGUE VIRUS EPITOPE VARIANTS
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