3602?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 115? ? ? Number 12? ? ? December 2005
Cross-reactive influenza virus–specific CD8+
T cells contribute to lymphoproliferation
in Epstein-Barr virus–associated
Shalyn C. Clute,1,2 Levi B. Watkin,1,2 Markus Cornberg,1 Yuri N. Naumov,1 John L. Sullivan,3,4
Katherine Luzuriaga,3,4 Raymond M. Welsh,1 and Liisa K. Selin1
1Department of Pathology, 2Program in Immunology and Virology, 3Department of Pediatrics, and 4Program in Molecular Medicine,
University of Massachusetts Medical School, Worcester, Massachusetts, USA.
There is a high degree of individual variation in disease severity
associated with human virus infections, and age is one of many
factors that can contribute to such variation. Childhood infec-
tion with EBV is often subclinical while the same infection is fre-
quently symptomatic in adolescents and adults and presents as
infectious mononucleosis (IM). The 3 classic criteria for IM diag-
nosis are the following: (a) lymphocytosis, the marked expansion
of lymphocyte numbers in the peripheral blood caused by the
proliferation of EBV-specific CD8+ T cells; (b) clinical symptoms
that include fever, pharyngitis, and lymphadenopathy; and (c) a
positive serologic test (1, 2). IM can vary in duration from a few
weeks to 6 months, and the symptoms can vary in severity (3).
Complications, such as pneumonia and fulminant hepatitis, are
more common in older adults and have been linked to the infil-
tration of activated T cells and EBV-infected B cells into these tis-
sues (4–6). When comparing IM and asymptomatic cases of acute
EBV infection, Silins et al. found that the magnitude of the CD8+
T cell response, not viral load, correlated with the presence of
disease (7). Furthermore, treatment of IM patients with antiviral
drugs, although decreasing viral load, did not have any effect on
the disease course (8, 9). These data suggest that a massive CD8+
T cell response can be counterproductive and mediate the disease
pathology. It is still unclear why this massive CD8+ T cell prolif-
eration occurs more frequently in older individuals.
Based on animal models of heterologous immunity that showed
that T cells specific to a previously encountered virus may enhance
immunopathology during a second, unrelated virus infection and
based on the increasing number of reports documenting virus-spe-
cific CD8+ T cell cross-reactivity, we hypothesized that cross-reac-
tive memory T cells specific to previously encountered pathogens
contribute to the lymphoproliferation characteristic of EBV-asso-
ciated IM (10–15). In support of this, there is well-documented
evidence that at least a proportion of the CD8+ T cells activated
by EBV can have alternative specificities for allogeneic MHC mol-
ecules, self peptides, and bacterial antigens (16–19). Testing this
hypothesis is challenging due to individual variation in HLA allele
expression, the history of infections, and the private specificity of
the responding T cell repertoire (20–22).
In order to optimize our chances of detecting whether cross-
reactive T cells were contributing to the EBV-induced CD8+ T cell
response during IM, we focused our studies on individuals with a
common HLA allele, A*0201, and their responses to a commonly
encountered virus, influenza A. The majority of the world’s popu-
lation, starting at a young age, are repeatedly infected with influ-
enza virus A. In almost all HLA-A2+?individuals, the CD8+ T cell
response to influenza A virus is focused on an immunodominant
epitope, M158–66, which is derived from the matrix protein (23).
This protein is well conserved among different virus strains, ensur-
ing the maintenance of M1-specific T cells in an individual’s mem-
Nonstandard?abbreviations?used: Cb, constant region of TCR b-chain; CDR3,
complementarity-determining region 3; EC50, effective concentration eliciting 50%
of the maximum response; IM, infectious mononucleosis; Jb, joining region of TCR
b-chain; LCMV, lymphocytic choriomeningitis virus; MIP-1b, macrophage inflamma-
tory protein-1b; Vb, variable region of TCR b-chain; VV, vaccinia virus.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 115:3602–3612 (2005).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
ory pool. While there is significant individual variation regarding
which EBV antigen(s) drive a dominant CD8+ T cell response, we
focused on the response to BMLF1280–288, an immunodominant
epitope within an early lytic protein that is also consistently rec-
ognized by all HLA-A2+ individuals during the acute response
(24–26). In this study, we detected a cross-reactive response with
specificity for these 2 dissimilar epitopes, influenza M1 and EBV-
BMLF1, in bulk T cell cultures and at the clonal level, and we dem-
onstrate that these cross-reactive cells participate in the character-
istic CD8+ T cell–mediated pathology observed during acute IM.
Maintenance of cross-reactive CD8+ T cells in the memory pools of healthy
donors. Using ex vivo tetramer staining, we found a small subset of
T cells in a healthy immune donor that recognized both influenza
M1 (GILGFVFTL) and EBV-BMLF1 (GLCTLVAML) epitopes (Fig-
ure 1A). In order to further verify this observation and enhance the
detection of cross-reactive T cells, we cultured CD8+ T cell lines in
the presence of either M1 or BMLF1 peptide, and their specificity
was then tested with an intracellular stain for the production of
IFN-γ. As expected, a large proportion (33%) of an M1-specific T
cell line derived from donor D-002 was able to produce IFN-γ fol-
lowing M1 stimulation (Figure 1B). However, a subset (3%) of this
same M1-specific line were able to produce a high level of IFN-γ
following BMLF1 stimulation (Figure 1B). The IFN-γ production
in response to BMLF1 was considered antigen specific because
stimulation with HLA-A2–presented peptides derived from HIV-
gag?or tyrosinase, a self antigen, resulted in very little IFN-γ pro-
duction. In the case of donor D-002, a putatively cross-reactive
subset of cells was also detected within the BMLF1-specific CD8+
T cell line (Figure 1C). Not only did the majority (59%) of the line
produce IFN-γ following BMLF1 stimulation, but at least 1% of the
T cell lines grown in the presence of 1 peptide can respond to stimulation with a second unrelated peptide. (A) CD8+ T cells were isolated ex vivo
from healthy donor D-002 and costained with M1- and BMLF1-loaded tetramers; 106 events were collected. (B and C) Fresh CD8+ T cell lines
derived from donor D-002 were grown for 3–4 weeks in the presence of (B) M1 peptide–pulsed or (C) BMLF1 peptide–pulsed T2 cells and then
stained intracellularly for the production of IFN-γ or MIP-1b following 5 hours of stimulation with various HLA-A2–restricted peptides at a 5 µM
final concentration. Percentages of CD8+ T cells producing each cytokine are shown. (D) Titration of peptide concentrations in an intracellular
IFN-γ assay using an M1-specific T cell line derived from donor D-002 demonstrated a slight difference in avidity for M1 versus BMLF1. Filled
triangles, tyrosinase; open circles, M1; and filled circles, BMLF1 stimulation.
3604?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
line produced a low level of IFN-γ following M1 stimulation. Table
1 summarizes the results of several intracellular IFN-γ stains on
both M1- and BMLF1-specific T cell lines derived from 8 healthy
donors with previous exposure to EBV and, presumably, influenza
A virus. Using this technique, we were able to detect cytokine pro-
duction to the heterologous peptide over background levels in cell
lines from 3 out of 8 healthy donors.
The observation that a greater proportion of the M1-specific
population compared with the BMLF1-specific population were
cross-reactive T cells in the blood (5% of M1-tetramer+ cells versus
0.3% of BMLF1-tetramer+ cells, Figure 1A) and in culture (3% of an
M1-specific line versus 1% of a BMLF1-specific line, Figure 1, B and
C) prompted us to compare the relative avidities of the cross-reac-
tive interaction with M1 versus BMLF1. To estimate TCR avidity,
we performed an intracellular IFN-γ assay, using a peptide titra-
tion, on an M1-specific T cell line derived from donor D-002. A
concentration of 5 × 10–8 M of M1 peptide compared with 10–7 M
of BMLF1 peptide resulted in about half of the maximum amount
of IFN-γ produced by this M1-specific T cell line (effective con-
centration eliciting 50% of the maximum response [EC50]) (Figure
1D). Thus, the avidity for these 2 epitopes was slightly different,
but both were within the avidity range previously reported for M1-
specific and BMLF1-specific T cell clones using IFN-γ production
or cytotoxicity as the readout (27, 28). Although the stronger M1
CD8+ T cell lines from multiple healthy donors responding to M1 and BMLF1 stimulation
Intracellular IFN-γ production
Influenza-M1–stimulated T cell linesA
EBV-BMLF1–stimulated T cell lines
Control T cell lines grown with unpulsed T2 cells
Intracellular MIP-1b production
Influenza-M1–stimulated T cell linesA
EBV-BMLF1–stimulated T cell lines
Control T cell lines grown with unpulsed T2 cells
PMA+ionomycin?M1?(influenza)?BMLF1?(EBV)? gag?(HIV)?Tyrosinase? No?peptide
AT cell lines were grown for a minimum of 3 weeks. Numbers indicate the percentage of CD8+ T cells that produce cytokine in response to the respective
stimuli. ND, not determined; NA, no cell line available. Bold numbers indicate that the percentage of a given T cell line, cultured with peptide, responding to
an unrelated peptide stimulation was greater than either the percentage of that same T cell line responding to negative control peptides or the percentage
of the control T cell line, cultured without peptide, responding to the same unrelated peptide stimulation.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
stimulus may have been more efficient at promoting the growth
of cross-reactive cells, it is likely that the cross-reactive population
growing in the M1-specific T cell line is composed of a distinctly
different subset of T cell clones than that growing in a BMLF1-
specific T cell line. In support of this, the same peptide titration
assay was performed using the BMLF1-specific T cell line, and this
resulted in a cross-reactive population with a much greater dis-
crepancy between the avidities for these 2 epitopes. Interestingly,
the EC50 in response to the cross-reactive M1 stimulation was
10–9 M compared with 5 × 10–7 M in response to BMLF1 stimu-
lation (Supplemental Figure 1; supplemental material available
online with this article; doi:10.1172/JCI25078DS1). Thus, the
cross-reactive cells grown in the presence of only BMLF1 peptide
had an even higher avidity for the M1 epitope than did the cross-
reactive cells grown in the presence of M1 peptide. The stimulating
antigen used in culture appeared to drive differences in the clonal
composition of M1- and BMLF1-specific T cell lines derived from
the same donor, and this likely explains the
lack of reciprocity in the frequency of the
cross-reactive cells found within them.
Cross-reactive stimulation also resulted in
the production of macrophage inflammato-
ry protein-1b (MIP-1b), an antiviral C-C (b)
chemokine. Nearly every cell of an M1-spe-
cific line derived from donor D-002 was able
to produce some level of MIP-1b following
stimulation with BMLF1 but, importantly,
not in response to stimulation with HIV-gag
or tyrosinase (Figure 1B). The production of
MIP-1b, therefore, appeared to be more sen-
sitive than the production of IFN-γ for the
measurement of cross-reactivity in lines that
had IFN-γ–producing cross-reactive T cells.
In cell lines that did not have IFN-γ–produc-
ing cross-reactive T cells, no MIP-1b was pro-
duced in response to cross-reactive stimula-
tion, suggesting that the protocol used here
did not support the outgrowth of cross-
reactive cells from the CD8+ T cell popula-
tions of donors D-046 and D-048 and that
the production of MIP-1b was specifically
induced by TCR engagement with antigen
(Table 1). Thus, MIP-1b production was?a
more sensitive measurement of cross-reactiv-
ity than IFN-γ production, but this required
cross-reactive cells to be present. There was
no detectable cross-reactive MIP-1b produc-
tion in the BMLF1-specific line derived from
donor D-002, which again likely reflects dif-
ferences in the clonotypic composition of
this BMLF1-driven T cell line compared with
the M1-driven T cell line (Figure 1C).
Breadth and quality of cross-reactivity revealed
through alternative techniques. The growth of
cross-reactive cells specific to both M1 and
BMLF1 improved when we cultured CD8+
T cells with both peptides simultaneously.
The frequency of cells that costained with
both M1- and BMLF1-loaded tetramers
increased to a range of 0.3–1.1%. The cross-
reactive cells that bound both tetramers were also able to respond
functionally to both epitopes. They produced MIP-1b, IFN-γ, and
TNF-α specifically following either M1 or BMLF1 stimulation,
but BMLF1 stimulation appeared to result in a more robust pro-
duction of all 3 cytokines (Figure 2A). However, a peptide titra-
tion assay revealed that these M1+ BMLF1+ cross-reactive T cells
actually had a slightly higher avidity for the M1 epitope. The EC50
of the cross-reactive response to the M1 peptide was 10–8 M com-
pared with 10–7 M in response to the BMLF1 peptide (Figure 3A).
The more robust functional response to BMLF1 initially observed
using a 5 µM concentration of peptide appeared to be an effect of
significant TCR downregulation, which decreased the sensitivity
of M1-tetramer binding.
The tetramer-based frequency of cross-reactive cells within this
T cell line was lower than the frequency based on function. The
subset of cells only able to bind the M1-loaded tetramer produced
MIP-1b and IFN-γ but very little TNF-α in response to BMLF1
Culturing with M1 and BMLF1 peptides simultaneously promotes the growth of cross-reac-
tive cells. A CD8+ T cell line derived from healthy donor D-002 was grown for 4 weeks in the
presence of both M1 and BMLF1 peptide–pulsed T2 cells. Cells were stimulated for 5 hours
with various peptides and then stained extracellularly with tetramers and intracellularly for
the production of MIP-1b, IFN-γ, and TNF-α. We gated (indicated by a bold box) on (A) the
percentage of CD8+ T cells that costained with both M1- and BMLF1-loaded tetramers, (B) the
percentage of CD8+ T cells that stained with only M1-loaded tetramer, and (C) the percent-
age of CD8+ T cells that stained with only BMLF1-loaded tetramer. We then assessed the
cytokine production of those cells in response to the following peptide stimulations: tyrosinase
(gray profiles), M1 (dotted lines), and BMLF1 (solid lines). The percentage of CD8+ T cells
producing each cytokine within the positive gate (horizontal lines) drawn is shown below each
of the corresponding histograms. (D) This T cell line was stained extracellularly with EBV-
BRLF1–loaded tetramer as a control.
3606?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
stimulation (Figure 2B). The cells that bound only BMLF1-load-
ed tetramer also showed some degree of functional cross-reac-
tivity. At least 35% of the BMLF1+ cells produced a low level of
MIP-1b in response to M1 stimulation although this cross-reac-
tive stimulation was not as efficient at inducing IFN-γ or TNF-α
production (Figure 2C). A separate peptide titration experiment
on this single BMLF1 tetramer–positive subset revealed that the
cross-reactive cells present in this subpopulation had a similar
avidity for the M1 and BMLF1 epitopes, where the EC50 was
5 × 10–7 M of either peptide (Figure 3). Overall, it would appear
that how a cross-reactive T cell interacts with its alternative
ligand is highly variable and that cross-reactive T cell popula-
tions are indeed heterogeneous. Hence, multiple techniques
are required to detect T cell cross-reactivity, including tetramer
staining and different functional assays. As shown here, TCR
avidity is an important factor to consider when detecting cross-
reactive T cell responses. An interaction between a cross-reactive
T cell and its alternative ligand may be too weak to stably bind
tetramer but may still be sufficient to induce a distinct hierarchy
of cytokine production. This is analogous to the observation
that certain non–cross-reactive influenza M1–specific clones
were unable to bind M1-loaded tetramers but produced IFN-γ
following M1 peptide stimulation (29).
Cross-reactivity at a clonal level. We next sought to clone these cross-
reactive cells from a polyclonal T cell line, using the experimental
design outlined in Figure 4A. Briefly, we allocated single T cells that
costained with both M1- and BMLF1-loaded tetramers into microw-
ells. The single cells were propagated for 2 weeks (referred to as
clones from here on) and then assessed for functional specificity. As
expected from the T cell line data, there was tremendous variability
in the functional characteristics of each clone in response to either
antigen. Of all the clones that grew, 8% produced IFN-γ following
stimulation with either M1 or BMLF1 (Figure 4B). Similarly, 11%
of the different clones analyzed killed both M1- and BMLF1-pulsed
target cells in a 51chromium release cytotoxicity assay (Figure 4C).
The number of functionally cross-reactive clones varied with the
technique used for their detection and reflected a similar ratio of
BMLF1-responders to M1-responders (4:1) as seen in the assessment
of IFN-γ production by the double-tetramer+ population within a
polyclonal T cell line (1.5:1) (Figure 2A). These results definitively
show that individual T cell clones can recognize and respond to both
M1 and BMLF1, 2 epitopes that share little sequence similarity.
Tetramer-defined subsets of cross-
reactive T cells differ in their avidity for
the 2 epitopes. A similar intracellular
IFN-γ assay was performed on the
same T cell line described in Figure
2, which had been grown in the pres-
ence of M1 and BMLF1 peptides for
4 weeks, using a titration of peptide
concentrations. Filled triangles,
tyrosinase; open circles, M1; and
filled circles, BMLF1 stimulation. We
assessed the IFN-γ production of
gated, tetramer-defined subsets of
the T cell line: (A) M1+ BMLF1+ and
(B) M1– BMLF1+.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
Cross-reactive cells participate in the lymphoproliferation that defines
IM syndrome. Since we were able to detect cross-reactive cells that
recognize M1 and BMLF1 in bulk culture and at a clonal level, we
next determined whether these cross-reactive cells participated in
the overzealous CD8+ T cell response that defines EBV-associated
IM. We noticed that, despite the large expansion of EBV-specific
cells, the frequency (as percentage of CD8+ T cells) of M1-specific
cells in IM patients (mean, 0.20%; range, 0.02–0.49%) was similar
to that in healthy influenza-immune donors (mean, 0.26%; range,
0.09–0.79%). The maintenance of a resting-state frequency sug-
gested that at least a subset of M1-specific cells were proliferating
in response to infection because non–cross-reactive memory cells
should be diluted out by the proliferation of virus-specific cells.
In fact, the average number of M1-specific cells (per ml of blood)
was a very significant 4-fold higher in IM patients (0.004 × 106 per
ml) compared with healthy donors (0.001 × 106 per ml) (P = 0.02)
(Figure 5A). Of 8 IM patients, 5 had a higher than average number
of M1-specific cells at presentation, and this number decreased
over the course of the infection with contraction kinetics similar
to the BMLF1 response and overall lymphoproliferation (Figure 5,
B–D). These observations suggested that the M1-specific popula-
tion within each of these 5 IM patients contained T cells that were
cross-reactive with an EBV-derived antigen.
We next costained freshly
isolated CD8+ T cells from IM
patients with M1- and BMLF1-
loaded tetramers. Double-posi-
tive cross-reactive cells were
prominent in 2 patients, E1101
and E1178 (Figure 6B and
Supplemental Figure 2). The
percentage as well as the total
number of cross-reactive cells
shifted with this active infec-
tion, including a considerable
increase at day 22 (0.003 × 106
M1+ BMLF1+ cells per ml of
blood) after presentation of
patient E1101’s clinical symp-
toms (Figure 6, A and B). This
translated to as many as 1/3 of
the T cells specific to the immu-
nodominant BMLF1 epitope
being cross-reactive with M1.
Fewer tetramer-defined cross-
reactive T cells were detected
during patient E1178’s infec-
tion, but there was a discern-
able increase in frequency at
days 12 and 34 (0.0002 × 106
M1+ BMLF1+ cells per ml of
blood) after presentation with
IM (Supplemental Figure 2).
These data support our hypoth-
esis that cross-reactive T cells
contribute to lymphoprolifera-
tion during IM. Although the
BMLF1-specific T cell popula-
tion represented only a minor
proportion (0.4–1.9%) of the
total CD8+ T cell pool in the blood, it remains possible that addi-
tional EBV-derived antigens simultaneously activate cross-reactive
memory T cells with specificity for antigens other than influenza
M1. Interestingly, both of these patients presented with symptoms
of IM but with differences in severity. Patient E1101, who had the
higher frequency of M1+ BMLF1+ cross-reactive cells, presented
with severe (grade 5 on a scale of 1–5) symptoms and signs of
IM, including fatigue, sweats, chills, sore throat, nausea, myalgia,
lymphadenopathy, pharyngitis, and stomatitis. Notably, reversion
of the CD4:CD8+ T cell ratio, a hallmark of an active viral infec-
tion, was not evident until day 34 after presentation. In contrast,
patient E1178, who had a lower frequency of cross-reactive cells,
presented with only moderate (grade 2–3) symptoms and signs
of IM, including fatigue, loss of appetite, and only mild hepato-
splenomegaly. For this patient, CD4:CD8+ reversion was observed
on days 0 and 6, with a second episode of reversion again at day 27
after presentation, suggesting fluctuations in disease course. More
patients will need to be studied to determine whether the correla-
tion of cross-reactivity with M1 and disease severity will hold, but
here we document high levels of cross-reactive T cells associated
with severe IM pathology.
Skewing of the M1-specific Vb17+ TCR repertoire during IM. The M1-
specific memory TCR repertoire is organized in a conserved pat-
Cross-reactive clones are heterogeneous in their response to M1 versus BMLF1. (A) An outline of the
experimental design used to clone cross-reactive T cells from healthy donor D-002 is shown. (B) CD8+ T
cells incubated for 20 hours with either M1-, BMLF1-, or tyrosinase-pulsed K562/HLA-A2 cells. The per-
centages shown represent the number of wells harboring cells that produced IFN-γ following stimulation
with both M1 and BMLF1 or following stimulation with only 1 of the peptides. (C) CD8+ T cells incubated for
8 hours with either M1-, BMLF1-, or tyrosinase-pulsed K562/A2 cells. The percentages shown represent
the number of wells harboring cells that killed both M1- and BMLF1-pulsed target cells or that killed only
1 target cell type in a 51chromium release assay. Data presented here accurately represent the trends
observed in 3 separate experiments.
3608?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
tern, as unique TCR subclones using the variable (Vb) gene family
Vb17 can be ordered in a distinct hierarchy based on their joining
gene (Jb) usage, where Jb2.7 dominates (used by 55–62% of sub-
clones), followed by Jb2.3 (used by 10–20% of subclones), and then,
often, Jb2.1, Jb2.5, Jb1.1, or Jb1.2 at lower, more variable frequen-
cies (Y.N. Naumov et al., unpublished observations). We investigat-
ed whether the M1-specific repertoires of E1101 and E1178 were
skewed from this pattern and thereby reflective of cross-reactive
TCR-mediated clonal expansions. Due to limited blood samples,
we were unable to sort and sequence the TCRs of the M1-specific
cells directly ex vivo. Rather, we generated M1-specific T cell lines
from these IM patients, and Vb analyses indicated that their M1-
specific repertoires were focused on the Vb17 family, similar to
that previously described for healthy individuals (data not shown)
(30–33). However, when we sequenced the Vb17+ subclones within
the cell lines derived from both patients, they did not follow the
highly conserved organizational pattern observed in healthy influ-
enza-immune donors. At day 22 after presentation, the time point
when the number of cross-reactive T cells was highest ex vivo, the
Jb2.3 family was overrepresented (30%) while the normally domi-
nant Jb2.7 family was vastly underrepresented (10%) within the
M1+ Vb17+ repertoire of patient E1101 (Figure 6, B and C). The
skewing of the repertoire was even more pronounced at day 165
(Jb2.3, 37%; Jb2.7, 3%), again a time when cross-reactive cells could
easily be detected ex vivo (Figure 6, B and C). When the M1-specific
population was presumably more stable at 1 year after presenta-
tion, the repertoire was still skewed from that typical of a resting
memory state but appeared to be slowly reverting, as the number of
subclones using Jb2.3 (25%) declined and those using Jb2.7 (14%)
were better represented (Figure 6C). Similarly, the M1-specific rep-
ertoire of patient E1178 was skewed during the acute phase of the
immune response to EBV but in a way that was different from that
of patient E1101. In the case of patient E1178, we found that the
Jb1.2 family was overrepresented within the M1+ Vb17+ repertoire
(50% at days 19 and 174), illustrating the point that a crossreactive
TCR repertoire may be unique to each individual and therefore not
easily predicted (Supplemental Figure 2). This perturbation in the
influenza M1–specific TCR repertoire of both patients with IM is
highly consistent with the concept that cross-reactive M1-specific
T cells are expanded in the host during acute EBV infection in an
antigen-driven manner by EBV-derived epitopes such as BMLF1.
We show here that cross-reactive T cells specific to a previously
encountered virus could be major contributors to the overzealous
CD8+ T cell response that defines IM. In 5 out of 8 patients, influ-
enza M1–specific CD8+ T cells participated in EBV-induced lym-
phoproliferation. Of these 5 patients, 2 had dramatically skewed
M1-specific TCR repertoires and increased levels of clearly identifi-
able, tetramer-defined, cross-reactive CD8+ T cells capable of rec-
ognizing the 2 dissimilar epitopes influenza M1 and EBV-BMLF1.
Based on our ability to culture these cross-reactive cells from 3 out
IM patients have an augmented number of M1-specific cells in their bloodstream. PBMCs were isolated from 8 healthy donors or from 8 patients
experiencing IM. Blood from IM patients was collected at various points after presentation with symptoms of IM. Please note that the number
of data points for each patient is variable. (A–C) CD8+ cells were first isolated from PBMCs and then immediately stained with tetramer. The
percentages of tetramer-positive cells were used to calculate the total number of either M1- or BMLF1-specific cells per ml of blood. (A) The
difference between the means of the 2 subject groups was determined to be statistically significant using an unpaired, 2-tailed Student’s t test.
#P = 0.02. Patient E1197 had an M1-specific memory population that grew out in culture but was undetectable ex vivo; therefore, this patient
was excluded from calculation of the mean. (D) PBMCs were used to costain with CD3 and CD8+ antibodies and calculate the total number of
CD8+ T cells per ml of blood.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
of 8 healthy donors with previous exposure to both viruses, these
cross-reactive cells are maintained in memory and their functional
responses to either antigen can include cytotoxicity and the produc-
tion of MIP-1b, IFN-γ, and TNF-α. Cross-reactive T cells may play a
major role in the development of IM, and the diversity of their func-
tions may contribute to the severity of the syndrome. These studies
examined only one cross-reactive population while it is likely that
infection with EBV, a virus with the potential to encode hundreds
of epitopes, could reactivate many memory T cell populations yet
undefined. As demonstrated here, the identification of cross-reac-
tive T cells can be complicated by their ability to recognize alterna-
tive peptides having little sequence similarity to their native ligand,
their strict growth requirements in vitro, and the sensitivity of the
different techniques used to detect them. These are all challenges
for future elucidations of individual cross-reactive T cell responses
and their potential impact on the outcome of IM.
Our work suggests that acute EBV infection activated influenza
M1–specific CD8+ T cells. These cross-reactive M1-specific T cells
are most likely memory cells for the following reasons: (a) by this
age, everyone is immune to influenza A virus; (b) almost all HLA-
A2+ individuals develop an M1-specific response; and (c) costaining
with M1- and BMLF1-loaded tetramers showed that these cross-
reactive cells brightly stained with the M1-loaded tetramer, sugges-
tive of a high avidity interaction typical of antigen-specific memory.
The activation of cross-reactive memory cells coincident with the
development of IM disease pathology is highly analogous to the
examples of heterologous immunity we have observed in mouse
models, where memory T cell responses to a prior infection with an
unrelated virus altered the host’s immune response to a subsequent
infection and caused a marked deviation in disease course (34).
It has recently been shown that acute HIV infection can up-
regulate the expression of activation markers such as CD38,
HLA-DR, and Ki67 on memory cells specific to influenza A virus,
EBV, and CMV, but the role of the TCR in this activation was not
determined (35). Our work suggests that acute EBV infection can
activate influenza-specific memory cells through a TCR-depen-
dent mechanism. The expansion of M1-specific memory cells was
evident in only 5 of 8 patients with IM despite the fact that all
probably had memory to M1 and all would have been influenced
by any cytokine-mediated, or bystander, activation. When pos-
sible, we also looked for the expansion of a second memory T cell
population, specific for CMV-pp65. Only 2 IM patients proved to
be CMV seropositive, E1155 and recent enrollee E1238. The fre-
quency of pp65-specific T cells in patients E1155 and E1238 was
low during massive, EBV-induced lymphoproliferation. At day 0,
E1155 and E1238 had a pp65-specific T cell frequency of 0.2% and
0.7% respectively while, by 41–50 days after presentation, those fre-
quencies climbed to 0.6% and 1.1% respectively. These data would
suggest that the pp65-specific memory populations of these 2
patients did not contain T cell clones cross-reactive with EBV and
were therefore initially diluted out by the extensive proliferation
of EBV-specific T cells.
For 2 of the 5 patients with higher M1 frequencies (E1101 and
E1178), we determined that BMLF1 was at least 1 of the EBV-
derived antigens recognized by cross-reactive T cells. Although
both patients shared this particular pattern of cross-reactivity,
their responses remained unique. Vb17+ subclones using Jb2.3
preferentially expanded in patient E1101 while those using Jb1.2
preferentially expanded in patient E1178. Thus, the M1-specific
TCR repertoire of both patients was notably skewed from that
known to be conserved among healthy HLA-A2+ individuals (Y.N.
Naumov et al., unpublished observations). These data are further
suggestive of antigen-driven clonal expansions because a bystand-
er activation mechanism would drive the expansion of all clones
and would maintain the conserved repertoire organization. The
fact that cross-reactive T cells specific for M1 and BMLF1 were
observed in 2 but not all patients is probably reflective of these
clonal differences, known as the private specificity of each indi-
vidual TCR repertoire. In support of this, cross-reactive T cell
responses involving lymphocytic choriomeningitis virus (LCMV)
and vaccinia virus (VV) showed that only 50% of VV-challenged
LCMV-immune mice mounted a strong response to a specific
Acute EBV infection augments the number of cross-
reactive cells that recognize M1 and BMLF1. CD8+ T
cells were isolated ex vivo from patient E1101 at vari-
ous time points after presentation with symptoms of IM.
(A) The total number of antigen-specific T cells per ml of
blood was calculated using the frequencies of tetramer-
positive cells. BMLF1+, tetramer positive; M1+ BMLF1+,
double-tetramer positive; ND, not determined because
the frequency was below the limit of detection using this
technique. (B) The percentages of CD8+ T cells staining
positive when costained with M1- and BMLF1-loaded
tetramers are shown. The number of events shown is
variable because the maximum number possible was col-
lected for each sample. (C) CD8+ T cells isolated at days
22, 165, and 349, were cultured for 3 weeks in the pres-
ence of M1 peptide–pulsed T2 cells. Following the RNA
isolation and cDNA synthesis of those T cell lines, the
CDR3b region of Vb17+ subclones was sequenced. The
pie charts illustrate the percentages of unique Vb17+ sub-
clones using each Jb family, where n = the total number
of unique subclones. The complete CDR3 sequences of
all the subclones analyzed are displayed in Supplemental
Table 1, structured according to Chothia et al. (57).
3610? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
LCMV epitope, NP205–213, and this was a function of the private
specificity of the LCMV memory TCR repertoire of each mouse
(22). The existence of T cell responses unique to the individual
adds to the complexity in resolving the importance of cross-reac-
tive responses in human infections.
Previous studies showed that the extent of T cell activation and
proliferation correlated with the severity of IM (7, 36). We suggest
that the magnitude of the T cell response represents the combined
efforts of cross-reactive memory and primary T cells. Our limited
clinical data indicate that the number of cross-reactive cells spe-
cific for M1 and BMLF1 may correlate with disease severity, but
further investigation is necessary to confirm this observation. The
severity of IM might be influenced by both the number of acti-
vated cross-reactive cells and their effectiveness at clearing virus.
There is recent evidence for the exacerbation of human disease by
the activation of cross-reactive influenza-specific memory CD8+ T
cells during acute HCV infection. Like EBV, acute HCV infection
is often asymptomatic, but when clinical symptoms manifest,
they are likely caused by the immune response. HCV encodes an
epitope, NS31073–1081, that can activate influenza NA231–239–spe-
cific memory cells (14). However, unlike EBV-BMLF1 and influ-
enza M1, which are only 33% similar in sequence, HCV-NS3 and
influenza NA are 78% similar in sequence. In congruence with our
observations during EBV infection, the activation of these cross-
reactive NA-specific memory cells enhanced the magnitude of
the CD8+ T cell response to HCV and resulted in severe disease
pathology (37). Despite strength in numbers, these HCV-spe-
cific T cells were unable to sufficiently clear the virus, and the
patients developed persistent HCV infections (37). Cross-reactive
cells may be inefficient at interacting with infected host cells due
to a low avidity for the alternative ligand, but they may still pro-
duce pathology-generating cytokines. Their presence may inter-
fere with the primary T cell response by preventing access to the
infected cells or changing patterns of T cell immunodominance,
thereby prolonging resolution of the infection.
Our work in animal models has suggested that cross-reactive
T cells can induce pathological conditions despite their ability
to clear virus. LCMV-specific memory cells lower the titer of
VV delivered intranasally but in doing so also alter the disease
pathology from pulmonary edema to bronchiolitis obliterans
(34, 38, 39). The cytokines, notably IFN-γ, secreted by these
activated memory cells may have played a major role in the
development of this immunopathology. In the present study,
we showed that cross-reactive cells specific to M1 and BMLF1
secreted several cytokines in a hierarchal pattern whereby most
secreted MIP-1b, fewer secreted IFN-γ, and even fewer secreted
TNF-α. Similar functional hierarchies were observed in studies
with freshly isolated HIV- or CMV-specific CD8+ T cells from
healthy donors with these persistent infections as well as during
the primary immune response to EBV in IM patients (40–42).
The mechanism behind T cell functional heterogeneity has been
extensively studied by varying the quantity of TCRs engaged and
by varying the quality of ligands used to engage them (28, 43–45).
Our work presents an opportunity to apply this knowledge to T
cell cross-reactivity involving 2 natural viral epitopes in order to
understand how cross-reactive T cells mediate the development
and severity of IM. Interestingly, both MIP-1b and TNF-α levels
are known to be elevated in the serum and tonsils of IM patients
compared with healthy controls (46, 47). MIP-1b can be readily
secreted because it is preformed and stored within human CD8+
T cells (48). Even a low avidity interaction may stimulate its
release from the cell. This is logical because MIP-1b broadens the
immune response by recruiting other immune cells to the site of
infection (49). The increase in number of activated immune cells
may enhance IM severity. Fewer of the cross-reactive cells secreted
TNF-α following peptide stimulation, suggesting that a higher
avidity interaction may be required to initiate its production.
However, an overall increase in the number of responding T cells
capable of secreting TNF-α at the site of infection could be harm-
ful to the host and promote the clinical symptoms of IM (50, 51).
In fact, the high production of TNF-α, possibly by cross-reactive
memory cells, has been implicated in the immunopathogenesis
associated with dengue hemorrhagic fever (52). Thus, depending
on the cross-reactive specificity pattern and private specificity of
the TCR repertoire, cross-reactive memory T cells activated by
EBV may function and modulate the disease outcome of each
individual very differently. Identifying these cross-reactive pat-
terns will be a challenge for the future.
In conclusion, our data suggest that cross-reactive memory T
cells participate in the massive lymphoproliferation that charac-
terizes EBV-associated IM and may influence disease severity. For
the purposes of this study, we focused on detection and activation
of M1-specific memory cells, but EBV, a virus with a large genome
that encodes numerous different proteins, has the potential to
generate epitopes reactive with many different memory T cell
populations. The cross-reactive pattern that emerges is influenced
both by an individual’s unique history of infection and by the pri-
vate specificity of the TCR repertoire responding to each of those
infections. Overall, this demonstration of cross-reactivity involv-
ing 2 immunodominant epitopes derived from 2 of the most com-
mon human viruses among people that share the most common
MHC class I haplotype in North America highlights the potential
importance of cross-reactive T cells in human disease states.
Subjects. Influenza A virus–immune patients between the ages of 18 and 23
with acute EBV infection were volunteers from the University of Massa-
chusetts (UMass) Student Health Services (Amherst, Massachusetts, USA).
HLA typing was performed using the Lymphotype Class I system (Biotest)
and an Olerup SSP kit (GenoVision). Acute EBV infection was confirmed
by a monospot test and the detection of capsid-specific IgM in patient sera.
Positive staining with HLA-A2 tetramers loaded with influenza M1 was
used as an indication that these individuals had been exposed to influenza
A virus in the past. Patients provided up to 8 blood samples (50 ml each)
starting at presentation with IM (day 0), then weekly for the following 6
weeks, and then again at 1 year. Healthy donors between the ages of 24 and
50 were volunteers from the research community at UMass Medical School
(Worcester, Massachusetts, USA). HLA status and immunity to EBV and
influenza A virus were assessed using monoclonal antibody (BB7.2; BD)
and tetramer stains, respectively. Previous exposure to EBV was confirmed
by the detection of capsid-specific IgG in donor sera. Donors provided up
to 3 blood samples (60 ml each). This study was approved by the Human
Studies Committee at UMass Medical School, and all subjects participat-
ing in our study gave informed consent.
Blood preparation and bulk T cell culture. PBMCs were isolated using Ficoll-
Paque plus (Amersham Biosciences) and were stained with anti-CD8+
microbeads before being positively selected using the Miltenyi Biotech
MACS system. CD8+ lymphocytes were plated at 2.5 × 105 per ml together
with peptide-pulsed (1 µM), irradiated (30 Gy) T2 cells (CRL-1992; ATCC)
at 5 × 104 per ml in 4 ml total volume per well of a 12-well plate. T cell lines
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
were fed media (AIM-V [Gibco; Invitrogen Corp.] supplemented with 14%
human AB serum [Nabi Biopharmaceuticals], 16% MLA-144 supernatant
[ref. 53], 10 U/ml rIL-2 [BD], 1% l-glutamine [Gibco; Invitrogen Corp.],
0.5% b-mercaptoethanol [Sigma-Aldrich], and 1% HEPES [HyClone]) every
3–4 days and were restimulated with T2 cells weekly.
HLA-A2–restricted peptides. The following peptides were synthesized to
greater than 90% purity by Biosource: EBV-BMLF1280–288 (GLCTLVAML),
influenza A virus-M158–66 (GILGFVFTL), tyrosinase (YMNGTMSQV),
and HIV-gag77–85 (SLYNTVATL).
MHC class I tetramers. A detailed description of the protocol used by the
tetramer facility at UMass Medical School has been previously published
(25). Tetramers were assembled using the above peptide sequences for EBV-
BMLF1 and influenza M1 and were conjugated to PE (Sigma-Aldrich),
APC (CALTAG Laboratories), or Quantum Red (Sigma-Aldrich). Tetramers
assembled with HIV-gag or tyrosinase (Immunomics) were used as negative
controls, and nonspecific staining was never observed.
Extracellular and intracellular staining. Cells were plated at 106 per well
and washed with staining buffer (PBS, 2% FCS, and 1% sodium azide).
Tetramers were incubated at room temperature for 40 minutes and were
washed off. Cells were either fixed with FACS Lysing Solution (BD) or per-
meabilized using Cytofix/Cytoperm (BD) according to the manufacturer’s
instructions. The following monoclonal antibodies were used: anti–IFN-γ
(B27; BD), anti–MIP-1b (D21-1351; BD), and anti–TNF-α (mAb11, eBio-
science). Isotype control antibodies did not stain positive.
T cell cloning. T cell?lines were costained in 2% FCS/PBS with tetramers
as described above. Double tetramer-positive?cells were sorted using BD
Vantage into 96-well plates at 1 cell per well. Each well contained 105 irra-
diated donor-specific CD4+ T cell blasts and 2 × 103 of a 1:1 mixture of
irradiated T2 cells pulsed with BMLF1 or M1 peptides. Clones were fed
media every 3–4 days and restimulated with T2 cells at day 7; their speci-
ficity was tested on day 14.
IFN-γ ELISPOT. Our protocol was adapted from a previously published
method using Mabtech reagents (54). The APCs were K562 cells stably
transfected with HLA-A2.1 and pulsed with 50 µM peptide for 3 hours
at 37°C, then washed of free peptide. Clones were tested using split-well
analysis; 5 µl of each clone was loaded per well per peptide-loaded cell type.
Plates were stained with streptavidin-horseradish peroxidase (Mabtech),
diluted 1:100, and Nova Red Substrate (Vector Laboratories) according to
the manufacturers’ instructions, then read manually.
51Chromium release assay. K562/HLA-A2.1 cells (54) served as the targets
and were pulsed with 100 µM peptide for 1 hour at 37°C and then for
an additional hour with 100 µCi 51Cr per 106 cells. Targets were washed
3 times and plated at 5 × 103 per well. Clones were tested using split-well
analysis; 30 µl of each clone was loaded per well per target type. Plates were
incubated for 8 hours at 37°C, and the supernatants were analyzed with a
MicroBeta TriLux scintillation counter (PerkinElmer).
Sequencing the TCR CDR3b. RNA was isolated from 106 cells derived from
T cell lines using TRIzol (Invitrogen Corp.) according to the manufacturer’s
instructions. Using 0.5–1 µg of RNA, cDNA synthesis was performed with
2 µM poly-T (Integrated DNA Technologies) and Superscript III reverse
transcriptase (Invitrogen Corp.) according to manufacturer’s protocol. The
cDNA was amplified with 1.6 µM each of Vb17-specific (nomenclature of
Arden et al.; ref. 55) and constant gene–specific (Cb-specific)?primers (56)
using the protocol recommended by Applied Biosystems with AmpliTaq
DNA polymerase. PCR products were ligated into the pCR4-TOPO vector
(Invitrogen Corp.) and were used to transform TOP10 chemically com-
petent cells (Invitrogen Corp.) according to the manufacturer’s protocol.
Individual colonies were picked for overnight cultures. DNA was isolated
with QIAprep miniprep kits (QIAGEN), and complementarity-determin-
ing region 3b (CDR3b) was sequenced at the UMass Nucleic Acid Facility
(Worcester, Massachusetts, USA) using universal primers.
We thank Rose Cicarelli, Robin Brody, and Joyce Pepe for their
technical assistance. This work is supported by a Worcester Foun-
dation grant, Deutsche Forschungsgemeinschaft fellowship CO
310/1-1, and NIH grants AI-49320, AI-42845, and DR-32520 and
immunology training grant 5 T32 AI-07349-16. The contents of
this publication are solely the responsibility of the authors and do
not represent the official view of the NIH.
Received for publication March 18, 2005, and accepted in revised
form October 4, 2005.
Address correspondence to: Liisa K. Selin, Department of Pathology,
Room S2-238, University of Massachusetts Medical School, 55 Lake
Avenue North, Worcester, Massachusetts 01655, USA. Phone: (508)
856-3039; Fax: (508) 856-0019; E-mail: email@example.com.
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