Antiviral T cell responses: Phalanx or multipronged attack?

Department of Medicine, Division of Hematology-Oncology, Weill Medical College, Cornell University, New York, NY 10021, USA.
Journal of Experimental Medicine (Impact Factor: 12.52). 07/2005; 201(12):1881-4. DOI: 10.1084/jem.20050928
Source: PubMed
ABSTRACT
Around 700 BCE, a new military formation called the phalanx was established in ancient Greece: a tight column of heavy infantry carrying long spears, or pikes, used in a single prong of attack. Later, in the battle of Marathon described by Herodotus, the Greeks learned the advantages of multipronged attacks, a strategy still used in modern warfare. Is the immune system similar in its approach to combatting pathogens or tumors?

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Vol. 201, No. 12, June 20, 2005 1881–1884 www.jem.org/cgi/doi/10.1084/jem.20050928
COMMENTARY
1881
Antiviral T cell responses: phalanx or
multipronged attack?
David N. Posnett, Manuel E. Engelhorn, Alan N. Houghton
Around 700 BCE, a new military formation called the phalanx was established
in ancient Greece: a tight column of heavy infantry carrying long spears,
or pikes, used in a single prong of attack. Later, in the battle of Marathon
described by Herodotus, the Greeks learned the advantages of multipronged
attacks, a strategy still used in modern warfare. Is the immune system similar
in its approach to combating pathogens or tumors?
Concrete evidence that a diverse, multi-
pronged T cell response is more effective
than a single-pronged response in con-
trolling viral infection in vivo in humans
is quite limited. Two recent papers in the
JEM
describe the T cell response to hu-
man cytomegalovirus (hCMV) and point
out that successful outcomes, with control
of viremia, are correlated with a more
polyclonal and diverse response (1, 2).
CD8
T cell responses to hCMV
hCMV infects over 50% of the human
population. Although hCMV encodes
200 gene products (3), the cellular
immune response is thought to be fo-
cused on two proteins, IE-1 and pp65.
80% of hCMV-specific CD8
T cells
are estimated to target these two pro-
teins (4), but with new epitopes being
discovered at an ever-increasing rate,
these figures may change. The CD8
T cell response is critical for mainte-
nance of clinical “latency.” Suppression
of CD8
T cell responses leads to viral
replication and disease, whereas adop-
tive transfer of hCMV-specific CD8
T cells results in reconstitution of effec-
tive cellular immunity (5).
pp65 is an abundant tegument pro-
tein produced as an early and late gene
product. It is considered the major target
of hCMV-specific cytolytic T lympho-
cytes (CTLs) based on classical cyto-
toxicity assays. Prior to MHC tetramer
technology and cytokine-based assays,
CTLs specific for IE-1 were not well
appreciated. IE-1 is an immediate early
gene product with a key role in trans-
activation of other viral genes. Several
hCMV gene products interfere with
MHC-I and MHC-II antigen presenta-
tion (6). pp65 itself blocks presentation
of IE-1 peptides via the MHC class
I pathway and inhibits expression of
genes associated with the induction of
interferon responses (3). It is therefore
possible that IE-1–specific responses re-
quire cross-presentation by an uninfected
cell to avoid the inhibitory effects of
pp65 (7). It has not been clear what the
biological role of IE-1– versus pp65-
specific responses might be, but the fact
that hCMV has evolved a strategy to
avoid IE-I–specific T cell responses
suggests an important role for these
cells in control of viral infection.
This conclusion was recently sup-
ported by a paper in the
JEM
by Bunde
et al. (1). These investigators examined
reactivation of latent viral infection in
immune-suppressed patients, which is a
major clinical problem in the field of
transplantation. In 27 transplant patients
on immunosuppressive drugs, they found
a correlation between an early CD8
T
cell response to IE-1 and protection
against hCMV disease. Those patients
that developed hCMV disease had CD8
T cell responses only to pp65 and some-
times lacked CD4
T cell responses to
pp65, IE-1, or both. The question of
diversity of the response was addressed
as a side issue. Although CD4
T cell
responses tended to be more diverse
in patients that did not develop dis-
ease, the difference was not statistically
significant.
Diversity of the hCMV-specific CD8
T cell response
In this issue of the
JEM,
Sacre et al. (2)
examined hCMV responses in several
groups of patients infected with both
HIV and hCMV in which the critical
distinction was whether or not the pa-
tients had active hCMV infection. Group
I consisted of HIV
patients with qui-
escent hCMV; group II were patients
being treated for hCMV infection who
either responded (group IIA) or required
continued treatment for greater than 5
years (group IIB), and group III were
patients with active hCMV infection.
The numbers of epitopes recognized
by CD8
T cells in Elispot assays, using
different pools of test epitopes, were
greater in those patients that controlled
hCMV infection: groups I and II. This
observation held true for both pp65
and IE-1 CD8
T cell responses. Group
IIA had greater IE-1–specific CD8
responses than group IIB, consistent
with the data from Bunde et al. (1),
suggesting that IE-1–specific responses
were protective.
Diversity of CD8
T cells in
other infections
Previous reports have indicated that
narrow CD8
T cell responses correlate
with viral persistence and that broad re-
sponses correspond to control and reso-
lution of viral infection. For instance,
in hepatitis C virus (HCV) infection,
broad and persistent CD8
T cell re-
sponses were associated with resolution
of viral infection, whereas weak and
D.N.P. is at the Department of Medicine, Division of
Hematology-Oncology, Weill Medical College,
Cornell University, New York, NY 10021.
M.E.E. and A.N.H. are at Swim Across America
Laboratory, Memorial Sloan-Kettering Cancer
Center, New York, NY 10021.
D.N.P., M.E.E., and A.N.H. are at The Immunology
Program, Graduate School of Medical Sciences,
Weill Medical College, Cornell University, New York,
NY 10021.
CORRESPONDENCE
D.N.P.: dposnett@mail.med.cornell.edu
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narrowly focused responses were seen
in patients with persistent infection (8).
CD4 T cell responses to several HCV
proteins were focused on an average of
10 epitopes in subjects with resolved in-
fection compared with
1 epitope in
those with persistent infection (9).
In primary HIV infection, it has been
known for some time that clonotypically
diverse CD8
T cell responses directed
at many, rather than few, epitopes are as-
sociated with a lower set point of viral
load and higher CD4
T cell counts
during the early phase of chronic infec-
tion, and therefore correlate with slow
disease progression (10–12).
Clonally restricted CD8
T cell ex-
pansions are frequently seen in chronic
persistent viral infections. Are they of
any utility to the host? In AIDS pa-
tients, they appear to be ineffectual in
controlling virus. Successful antiviral
therapy is associated with resolution of
these expanded clonotypes, suggesting
that the continued presence of replicat-
ing virus was driving the clonal expan-
sions (13). Similarly, massive expan-
sions of individual CD8
T cell clones
specific for hCMV have been observed
in elderly patients with hCMV infec-
tion where they appear to be ineffec-
tual and are associated with poor im-
mune function and possibly decreased
survival (4).
Different levels of T cell diversity
Studies that use only peptide pools to
quantify numbers of recognized epi-
topes, such as the papers discussed
above (1, 2), fail to assay for clonal T
cell receptor (TCR) diversity among T
cells that react to the same peptide–
MHC (pMHC) complex. Whether this
type of diversity is also important for
control of virus is not yet clear, al-
though analysis of CDR3 lengths in
TCRs from HIV-specific T cells does
support this conclusion (10).
Aged individuals have a more re-
stricted T cell repertoire, due to expan-
sion of memory clones and perhaps di-
minished thymic output of naive T
cells. Their immune responses may be
less heterogeneous on two levels: fewer
numbers of epitopes recognized and
less diversity of TCRs that recognize
individual pMHC complexes. Aged in-
dividuals have a decreased ability to
fend off infections, and one of several
proposed reasons may be this decrease
in T cell diversity.
Children with congenital hCMV
infection also have hCMV-specific
CD8
T cell responses targeted to pp65
and IE-1 (14). However, it is not yet
clear whether diversity of the T cell re-
sponse at the level of recognition of
larger numbers of epitopes is more typi-
cal of neonatal as opposed to adult
CD8
T cells. At the level of TCR di-
versity to a single epitope, the neonatal
response to an immunodominant matrix
peptide of influenza is very diverse using
many different TCRs. However, with
aging (and repeated exposures to influ-
enza), the response to the same pMHC
is dominated by TCR V
17 CD8
T
cells, such that depletion of this subset
abrogates the in vitro response to the in-
fluenza peptide (15). It is unclear how
this observation relates to the ability to
deal with influenza infection.
Is diverse better?
Does heterogeneity at the level of T
cell clones defined by TCR diversity
really matter? This question has been
addressed by Nikolich-Zugich and co-
workers (16). In mice, greater than
90% of CD8
T cell responses specific
for the immunodominant epitope gly-
coprotein B (gB498-505) of herpes
simplex virus (HSV)-1 use V
10 or
V
8 TCRs. The response is less vigor-
ous and less diverse in C57BL/6 (B6)
mice than in coisogenic bm-8 (B6.C-
H-2
bm8
) mice, which differ only by a
few amino acid residues in the peptide
binding groove of the MHC-I K
b
mol-
ecule. B6 mice succumb to viral infec-
tion at 25-fold lower doses of virus
than bm-8 mice (16). When diversity
was experimentally reduced, by elimi-
nating V
8 cells with an antibody, re-
sistance to infection was further re-
duced. Interestingly, the less diverse
CD8
T cell response in B6 mice was
also characterized by lower avidity in
MHC tetramer decay assays, which
measure the off-rate of the TCR–
pMHC interaction. The authors sug-
gested that a more diverse T cell re-
sponse provides a broader pool of
clones from which higher avidity CTLs
are more likely to be recruited. In old
mice that had a diminished repertoire
among CD8
T cells, particularly
within the V
8 family due to sponta-
neous CD8
T cell clonal expansions,
there were reduced numbers of MHC
tetramer
cells, undetectable antiviral
lytic function, and lowered resistance
to challenge with HSV-1 (17).
The clinical data discussed above
have only provided associations, and it
is conceivable that lack of a diverse re-
sponse is not the cause but rather the
result of uncontrolled viral infection.
Indeed, over time persistent viral anti-
gens are likely to cause an increasingly
focused T cell response dominated by
only a few clones. However, the ex-
periments of Nikolich-Zugich and col-
leagues (16) clearly show that in their
system lack of a diverse response can
result in decreased protection against a
lethal viral infection.
Relevance to tumor immunology
The diversity of the CD8
T cell re-
sponse is also relevant to cancer immu-
nity. Tumor-infiltrating antigen-spe-
cific CD8
T cells that arise with
tumor progression and T cells that are
stimulated after vaccination with
MAGE or Melan-A antigens are di-
verse. However, it has been assumed
that high avidity T cells (for example,
those that stain brightly with MHC tet-
ramers), which have superior antitumor
activity when tested in vitro (18), are
sufficient for tumor immunity. The di-
versity of the CD8
response to an ar-
tificial tumor antigen was decreased in
old compared with young mice (19),
but a careful study correlating the de-
gree of diversity of the CD8
response
and the efficiency of in vivo tumor
killing is still needed.
Promoting diversity
The arguments in favor of a diverse T
cell response have been made in previ-
ous papers (20) and are listed in Table
I. Side stepping the effects of the escape
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COMMENTARY
mutations in immunodominant epi-
tope, a favored trick of rapidly mu-
tating viruses such as HIV or SIV, is
one argument. Others pertain to avoid-
ing holes in the T cell repertoire that
arise due to ageing or to clonal anergy
or exhaustion. As discussed above, a
broader repertoire may provide choices
that allow for the selection of higher
avidity clones.
What are the mechanisms that pro-
mote a diverse response? Allison and
colleagues have suggested that the in-
hibitory receptor CLTA-4 serves to at-
tenuate extensive expansion of individ-
ual dominant clones, thereby allowing
other clones to expand and partici-
pate in the response. This attenuation
mechanism may ensure a polyclonal re-
sponse (21). In support of this model,
CTLA-4 expression has been shown to
increase as a function of the number of
cell divisions and high expression levels
of CTLA-4 correlate with inhibition of
clonal expansion. Cytokines might also
broaden the CD8
T response. Al-
though the mechanism is unknown,
exogenous IL-7 administered after im-
munization boosted the response to a
subdominant epitope of the experi-
mental antigen H-Y (22). If diversity is
truly important, there must be several
physiological mechanisms to promote
such a response. These mechanisms re-
main to be fully explored.
Practical applications
Assuming that a multipronged, diverse
CD4
and CD8
T cell response is a
good thing, how might this notion af-
fect future strategies for immunizations?
HIV vaccines are already being de-
signed that include many epitopes from
several genes and many peptide variants
(to cover all possible viral clades). But
this approach remains educated guess-
work, since the exact epitopes pre-
sented in a given patient, and therefore
the breadth of the immunization, are
unknown. To obtain a broad immune
response, additional strategies have been
proposed. Cytokines are being evalu-
ated as adjuvants (22). Engineered im-
munogens incorporating multiple het-
eroclitic peptides that cover an array
of immunodominant and subdominant
epitopes are being used successfully in
vaccine models. Alternatively, tumor-
derived RNA transcribed to cDNA can
be used for immunization, providing a
source of multiple antigens (23), and
virus replicase can be used to amplify
multiple RNA species in “replicon”
vectors (23, 24).
We conclude that the concept of
the phalanx as a mode of attack for the
immune system is outdated. Studies of
the natural immune response to com-
mon viruses like hCMV (1, 2) can
teach us all some important lessons re-
garding the advantages of diverse im-
mune responses. They provide a ratio-
nal basis for vaccines that produce
broad immune responses. This has spe-
cial relevance for the elderly, who need
vaccines most urgently and are least
likely to respond with an efficient and
broad response.
REFERENCES
1. Bunde, T., A. Kirchner, B. Hoffmeister, D.
Habedank, R. Hetzer, G. Cherepnev, S.
Proesch, P. Reinke, H.D. Volk, H. Lehm-
kuhl, and F. Kern. 2005. Protection from
cytomegalovirus after transplantation is cor-
related with immediate early 1–specific CD8
T cells.
J. Exp. Med.
201:1031–1036.
2. Sacre, K., G. Carcelain, N. Cassoux, A.-M.
Fillet, D. Costagliola, D. Vittecoq, D.
Salmon, Z. Amoura, C. Katlama, B. Autran,
and the RESTIMOP and ALT study
groups. 2005. Repertoire, diversity and dif-
ferentiation of specific CD8 T cells are asso-
ciated with immune protection against hu-
man cytomegalovirus disease.
J. Exp. Med.
201:1999–2010.
3. Britt, W.J., and S. Boppana. 2004. Human
cytomegalovirus virion proteins.
Hum. Im-
munol.
65:395–402.
4. Moss, P., and N. Khan. 2004. CD8(+)
T-cell immunity to cytomegalovirus.
Hum.
Immunol.
65:456–464.
5. Walter, E.A., P.D. Greenberg, M.J. Gilbert,
R.J. Finch, K.S. Watanabe, E.D. Thomas,
and S.R. Riddell. 1995. Reconstitution of
cellular immunity against cytomegalovirus
in recipients of allogeneic bone marrow by
transfer of T-cell clones from the donor.
N.
Engl. J. Med.
333:1038–1044.
6. Basta, S., and J.R. Bennink. 2003. A survival
game of hide and seek: cytomegaloviruses
and MHC class I antigen presentation path-
ways.
Viral Immunol.
16:231–242.
7. Tabi, Z., M. Moutaftsi, and L.K. Bo-
rysiewicz. 2001. Human cytomegalovirus
pp65- and immediate early 1 antigen-spe-
cific HLA class I-restricted cytotoxic T cell
responses induced by cross-presentation of
viral antigens.
J. Immunol.
166:5695–5703.
8. Lauer, G.M., E. Barnes, M. Lucas, J. Timm,
K. Ouchi, A.Y. Kim, C.L. Day, G.K. Rob-
bins, D.R. Casson, M. Reiser, G. Dusheiko,
T.M. Allen, R.T. Chung, B.D. Walker, and
P. Klenerman. 2004. High resolution analy-
sis of cellular immune responses in resolved
and persistent hepatitis C virus infection.
Gastroenterology.
127:924–936.
9. Day, C.L., G.M. Lauer, G.K. Robbins, B.
McGovern, A.G. Wurcel, R.T. Gandhi,
R.T. Chung, and B.D. Walker. 2002.
Broad specificity of virus-specific CD4+
T-helper-cell responses in resolved hepatitis
C virus infection.
J. Virol.
76:12584–12595.
10. Pantaleo, G., J.F. Demarest, T. Schacker, M.
Vaccarezza, O.J. Cohen, M. Daucher, C.
Graziosi, S.S. Schnittman, T.C. Quinn, G.M.
Shaw, L. Perrin, G. Tambussi, A. Lazzarin,
R.P. Sekaly, H. Soudeyns, L. Corey, and A.S.
Fauci. 1997. The qualitative nature of the pri-
mary immune response to HIV infection is a
prognosticator of disease progression indepen-
dent of the initial level of plasma viremia.
Proc.
Natl. Acad. Sci. USA.
94:254–258.
11. Edwards, B.H., A. Bansal, S. Sabbaj, J.
Bakari, M.J. Mulligan, and P.A. Goepfert.
2002. Magnitude of functional CD8+
T-cell responses to the gag protein of hu-
man immunodeficiency virus type 1 corre-
lates inversely with viral load in plasma.
J.
Virol.
76:2298–2305.
12. Dalod, M., M. Dupuis, J.C. Deschemin, D.
Sicard, D. Salmon, J.F. Delfraissy, A. Venet,
M. Sinet, and J.G. Guillet. 1999. Broad, in-
tense anti-human immunodeficiency virus
(HIV) ex vivo CD8(+) responses in HIV
type 1-infected patients: comparison with
anti-Epstein-Barr virus responses and changes
during antiretroviral therapy.
J. Virol.
73:
7108–7116.
13. Gorochov, G., A.U. Neumann, C. Parizot,
T. Li, C. Katlama, and P. Debre. 2001.
Down-regulation of CD8+ T-cell expan-
sions in patients with human immunodefi-
Table I.
Arguments in favor of a multipronged attack
Reference
1. Prevents escape mutants in immunodominant epitopes of SIV and HIV
(and other viruses) 25–28
2. Creates choices for the host to select the highest avidity TCRs 20
3. Avoids holes in the repertoire 20
4. Provides backup in case of clonal exhaustion or clonal anergy 29
20050928 Page 1883 Thursday, June 9, 2005 3:04 PM
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PHALANX OR MULTIPRONGED ATTACK | D.N. Posnett, M.E. Engelhorn, and A.N. Houghton
1884
ciency virus infection receiving highly active
combination therapy.
Blood.
97:1787–1795.
14. Gibson, L., G. Piccinini, D. Lilleri, M.G.
Revello, Z. Wang, S. Markel, D.J. Dia-
mond, and K. Luzuriaga. 2004. Human cy-
tomegalovirus proteins pp65 and immediate
early protein 1 are common targets for
CD8+ T cell responses in children with
congenital or postnatal human cytomegalo-
virus infection.
J. Immunol.
172:2256–2264.
15. Lawson, T.M., S. Man, S. Williams, A.C.
Boon, M. Zambon, and L.K. Borysiewicz.
2001. Influenza A antigen exposure selects
dominant Vbeta17+ TCR in human CD8+
cytotoxic T cell responses.
Int. Immunol.
13:
1373–1381.
16. Messaoudi, I., J.A. Guevara Patino, R. Dy-
all, J. LeMaoult, and J. Nikolich-Zugich.
2002. Direct link between mhc polymor-
phism, T cell avidity, and diversity in im-
mune defense.
Science.
298:1797–1800.
17. Messaoudi, I., J. Lemaoult, J.A. Guevara-
Patino, B.M. Metzner, and J. Nikolich-
Zugich. 2004. Age-related CD8 T cell
clonal expansions constrict CD8 T cell rep-
ertoire and have the potential to impair im-
mune defense.
J. Exp. Med.
200:1347–1358.
18. Dutoit, V., V. Rubio-Godoy, P.Y. Dietrich,
A.L. Quiqueres, V. Schnuriger, D. Rimoldi,
D. Lienard, D. Speiser, P. Guillaume, P. Ba-
tard, J.C. Cerottini, P. Romero, and D. Val-
mori. 2001. Heterogeneous T-cell response to
MAGE-A10(254-262): high avidity-specific
cytolytic T lymphocytes show superior antitu-
mor activity.
Cancer Res.
61:5850–5856.
19. Fang, L., D. Yarilin, J.R. Valiando, A.
Ronco, M.E. Weksler, P. Szabo, and D.N.
Posnett. 2002. Tumor antigen drives a per-
sistent oligoclonal expansion of CD8
T
cells in aged mice.
Eur. J. Immunol.
32:
1650–1658.
20. Nikolich-Zugich, J., M.K. Slifka, and I.
Messaoudi. 2004. The many important fac-
ets of T-cell repertoire diversity.
Nat. Rev.
Immunol.
4:123–132.
21. Egen, J.G., M.S. Kuhns, and J.P. Allison.
2002. CTLA-4: new insights into its biolog-
ical function and use in tumor immunother-
apy.
Nat. Immunol.
3:611–618.
22. Melchionda, F., T.J. Fry, M.J. Milliron,
M.A. McKirdy, Y. Tagaya, and C.L. Mack-
all. 2005. Adjuvant IL-7 or IL-15 over-
comes immunodominance and improves
survival of the CD8(+) memory cell pool.
J.
Clin. Invest.
115:1177–1187.
23. Boczkowski, D., S.K. Nair, D. Snyder, and
E. Gilboa. 1996. Dendritic cells pulsed with
RNA are potent antigen-presenting cells in
vitro and in vivo.
J. Exp. Med.
184:465–472.
24. Gilboa, E., and J. Vieweg. 2004. Cancer im-
munotherapy with mRNA-transfected den-
dritic cells.
Immunol. Rev.
199:251–263.
25. Barouch, D.H., J. Powers, D.M. Truitt,
M.G. Kishko, J.C. Arthur, F.W. Peyerl,
M.J. Kuroda, D.A. Gorgone, M.A. Lifton,
C.I. Lord, V.M. Hirsch, D.C. Montefiori,
A. Carville, K.G. Mansfield, K.J. Kunstman,
S.M. Wolinsky, and N.L. Letvin. 2005. Dy-
namic immune responses maintain cytotoxic
T lymphocyte epitope mutations in trans-
mitted simian immunodeficiency virus vari-
ants.
Nat. Immunol.
6:247–252.
26. Evans, D.T., D.H. O'Connor, P. Jing, J.L.
Dzuris, J. Sidney, J. da Silva, T.M. Allen, H.
Horton, J.E. Venham, R.A. Rudersdorf, T.
Vogel, C.D. Pauza, R.E. Bontrop, R. De-
Mars, A. Sette, A.L. Hughes, and D.I. Wat-
kins. 1999. Virus-specific cytotoxic T-lym-
phocyte responses select for amino-acid
variation in simian immunodeficiency virus
Env and Nef.
Nat. Med.
5:1270–1276.
27. Barouch, D.H., J. Kunstman, M.J. Kuroda,
J.E. Schmitz, S. Santra, F.W. Peyerl, G.R.
Krivulka, K. Beaudry, M.A. Lifton, D.A.
Gorgone, D.C. Montefiori, M.G. Lewis,
S.M. Wolinsky, and N.L. Letvin. 2002.
Eventual AIDS vaccine failure in a rhesus
monkey by viral escape from cytotoxic T
lymphocytes.
Nature
415:335–339.
28.
Price, D.A., S.M. West, M.R. Betts, L.E.
Ruff, J.M. Brenchley, D.R. Ambrozak, Y.
Edghill-Smith, M.J. Kuroda, D. Bogdan, K.
Kunstman, N.L. Letvin, G. Franchini, S.M.
Wolinsky, R.A. Koup, and D.C. Douek.
2004. T cell receptor recognition motifs
govern immune escape patterns in acute SIV
infection.
Immunity.
21:793–803.
29. Welsh, R.M., and J.M. McNally. 1999. Im-
mune deficiency, immune silencing, and
clonal exhaustion of T cell responses during
viral infections.
Curr. Opin. Microbiol.
2:382–
387.
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