Review

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Cytolytic CD4+ Tcells in

viral immunity
Expert Rev. Vaccines 9(12), 14531463 (2010)

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Damien Z Soghoian1
and Hendrik Streeck1
Ragon Institute of MGH, MIT and
Harvard Massachusetts General
Hospital, Harvard Medical School
Building 149, 13th Street, 5th floor,
#5217, Charlestown, Boston,
MA02129, USA

Author for correspondence:

Tel.: +1 617 726 3167
Fax: +1 617 726 5411
[email protected]
1

It is generally believed that the role of CD4 + Tcells is to coordinate the different arms of the
adaptive immune system to shape an effective response against a pathogen and regulate
nonessential or deleterious activities. However, a growing body of evidence suggests that
effector CD4 + Tcells can directly display potent antiviral activity themselves. The presence of
cytolytic CD4 + T cells has been demonstrated in the immune response to numerous viral
infections in both humans and in animal models and it is likely that they play a critical role in
the control of viral replication invivo. This article describes the current research on virus-specific
cytolytic CD4 + Tcells, with a focus on HIV-1 infection and the implications that this immune
response has for vaccine design.
Keywords : cytolytic CD4 + Tcells HIV vaccines viral infection

The concept that Tcells can play a helper role

dates back to the 1960s, when it was established
that thymocytes synergized with bone marrow cells to facilitate the production of anti
bodies [1,2] . The subsequent development of
mouse monoclonal antibodies to lymphocyte
antigens allowed for the further isolation of this
helper activity to CD4 + Tcells, which were also
recognized to provide helper signals to cytolytic
CD8 + Tcells [3,4] . However, it was noticed by
some groups that CD4 + Tcells (first as classII
reactive cells) could themselves exhibit cytotoxic
activity[5] . Although this activity was recognized
to only contribute a minor amount to the overall
cytolytic potential of Tcells in the blood, cytolytic CD4 + T-cell lines and clones could none
theless readily be isolated [6,7] . At the time, it was
suggested that this activity was a side effect of the
conditions required to culture Tcells invitro[8] .
For the most part, interest in cytolytic CD4 +
Tcells waned as immunologists focused on their
helper functions. The Th1 and Th2 subsets were
quickly established and became the paradigm for
defining T-cell help in the ensuing years [9] .
More recently, the CD4 + subset lineages have
been redefined, not only on the basis of their
effector functions, but also by the expression
of characteristic transcription factors. By now,
seven different CD4 + T-cell subsets have been
defined in humans but it is likely that more will
be distinguished in the future. Moreover, recent
advances in CD4 + T-cell research have begun to
revise the concept of immunomodulatory CD4 +
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10.1586/ERV.10.132

T-cell help and have indicated that a direct antiviral activity by these cells may be critical for
pathogen clearance. However, in the case of
HIV-1 infection, where activated CD4 + Tcells
are the main targets, the importance of CD4 +
Tcells in the control of the virus is still contro
versial [10] . The induction of cytolytic CD4 +
T-cell responses has therefore been seen with
great skepticism. This article will focus on the
current evidence and knowledge of the cytolytic
properties of CD4 + Tcells and will evaluate their
potential role in vaccine design.
CD4 + Tcells as direct effectors

The primary function of CD4 + helper Tcells is

to direct and focus immune responses to maximize antipathogenic processes, while suppressing
nonessential immune responses. This modulatory capacity of CD4 + helper Tcells is central to
the proper functioning of the immune system.
However, recent achievements in CD4 + T-cell
research have changed our thinking regarding
these cells dramatically. While it was commonly believed that Th1 cells provide help to
CD8 + Tcells and Th2cells generally provide
help to Bcells, this dichotomy has been revised
by the description of additional subsets (Th9,
Th17, Th21, T follicular helper [TFH] cells and
Tregulatory cells [Tregs]), each of which plays
a distinct role in the overall immune response.
Upon presentation of viral peptides by antigenpresenting cells, CD4+ Thelper cells become activated, secrete cytokines and clonally expand. The

2010 Expert Reviews Ltd

ISSN 1476-0584

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Soghoian & Streeck

differentiation of naive CD4 + T helper cells into distinct subsets

occurs during the progression of an infection and depends fundamentally on the effect that the infection has on the antigenpresenting cell. The affinity and strength of the T-cell receptor
major histocompatability complex (MHC) interaction, as well as
the cytokine/chemokine milieu present during initial CD4 + T-cell
activation, greatly influence subsequent T-cell differentiation[11] .
The CD4 + subsets that result from this differentiation are defined
primarily on the basis of their ability to secrete different cytokines
and the expression of specific transcriptional factors. Th1 cells are
characterized by the expression of the transcription factor Tbet
and the production of their cardinal cytokine/chemokine, IFN-g,
along with TNF-a and IL2 [12] . Th2 cells, on the other hand,
produce IL4, IL5 and IL10 under the control of the transcription factor GATA3 and preferentially act on the humoral arm of
the adaptive immune system. These cells have been shown to be
important for the induction of antibody class-switching to the
IgE and certain IgG isotypes [13] . TFH cells have been recently
described to be central for B-cell proliferation, maturation and the
induction of somatic hypermutation within the germinal center of
B-cell follicles. TFH cells are defined by the transcription factor
B-cell lymphoma 6 (BCL-6) and surface expression of CXCR5 and
inducible costimulator (ICOS) [14] . However, both surface antigens
are also present on non-TFH cells, demonstrating the overlap of
effector potential with other CD4 + subsets. The functional properties of TFH cells in humans are currently not well understood;
TFH cells in general produce the cytokine IL21, which functions
to stimulate Bcells but may also act in an autocrine fashion to
amplify TFH activity [15] . The production of IL21 is especially
interesting as IL21 has been linked to important antiviral helper
functions for CD8 + Tcells [1618] . It has been suggested that, in
addition to TFH cells, a separate CD4 + T-cell subset primarily
secreting IL21 may exist to mediate these antiviral activities [19] .
Th17 CD4 + Tcells, which produce IL17, have also been suggested
to secrete IL21. Th17 cells have been defined on the basis of the
transcription factor RAR-related orphan receptor gt (ROR-gt) and
are induced in the presence of IL6, TGF-b and IL1b [2022] . IL17
attracts and activates neutrophils and facilitates proinflammatory
responses from various other cell types (e.g., endothelial cells). By
contrast, CD4 + Tregs have been described as a CD4 + subset with
suppressive activity on innate and adaptive immunity and have
been shown to be enriched in chronic viral infections. Tregs are
defined by the coexpression of the zinc-finger transcription factor
forkhead box P3 (FoxP3) and the IL-2 receptor (CD25). Although
it is known that Tregs can be induced through the presence of
TGF-b (iTregs), they also can be selected in the thymus in the
early stages of differentiation (natural Tregs). They are believed
to act by a cellcell contact-mediated mechanism that does not
depend on soluble factors such as TGF-b or IL10, although these
factors might have an impact invivo [23,24] . Interestingly, it is also
notable that CD4 + CD25 + Tcells have been linked to a cytolytic
phenotype and it has been speculated that, despite their suppressive
nature, Tregs have a second, cytolytic function. However, cytolytic
CD4 + Tcells might also represent their own, less well described,
CD4 + T-cell subset.
1454

Cytolytic CD4 + Tcells

Very little is known about the phenotype, function and transcriptional profile of cytolytic CD4 + Tcells. Although cytotoxic
activity is traditionally thought to be the role of natural killer
(NK) cells or CD8 + cytotoxic T lymphocytes (CTLs), there is
no evidence to suggest that these cells are linked to the differentiation of cytolytic CD4 + Tcells. Although controversy persists
about their relevance, cytolytic CD4 + Tcells have been implicated in the control of a variety of persistent viral infections,
such as EpsteinBarr virus (EBV), hepatitis C virus (HCV) and
HIV-1 [2527] . Moreover, cytolytic killing of virally infected cells
by CD4 + Tcells has been also recently observed invivo in the
lymphochoriomeningitis virus (LCMV) mouse model [28] . It is
therefore possible that these cells comprise a novel CD4 + subset
with a unique lineage and functionality. This underestimated
direct effector activity of CD4 + Tcells raises questions about their
nature, role and the mode of induction of cytotoxicity.
Phenotype

Early studies describing the presence of cytolytic CD4 + Tcells

in the context of influenza and poliovirus categorized these cells
broadly as belonging to the Th1 subset. This conclusion was
based on the cytokine secretion profiles of cytolytic CD4 + clones
as well as the observation thatcells polarized invitro to the Th1
phenotype could exert cytolytic activity in killing assays, in contrast to Th2 cells [29,30] . These findings have been extended to
other viruses, such as West Nile Virus and EBV[3133] , as well as
to TB infection [34] and cancer [35] . However, the functional profile of cytolytic CD4 + Tcells appears to be distinct from CD4 +
Tcells with a Th1 phenotype. Moreover, while classical Th1
cells show the ability to secrete IL2, the majority of cytolytic
CD4 + Tcells lose this effector function [36] . By contrast, Th1
cells show higher levels of the costimulatory molecules CD28
and CD27 after initial antigenic stimulation, while cytolytic
CD4 + Tcells lack expression of these markers [26,36] . Similar
differences have been observed for the expression of the integrin
a chains CD11a and CD11b, which are upregulated on CD4 +
Tcells with cytolytic effector potential [36] . This marker profile
is suggestive of a terminally differentiated effector cell phenotype; indeed, the expression patterns of other markers associated
with such a phenotypesuch as CD57high and CCR7loware
found on perforin or granzyme-positive CD4 + Tcells [36,37] .
Interestingly, perforin or granzyme-expressing CD4 + Tcells
with this terminally differentiated phenotype are present even
in healthy individuals and are markedly expanded in those with
chronic viral infections [36] .
Further studies have suggested that cytolytic CD4 + Tcells may
also express molecules that are normally found on NK cells such
as NKG2D, KIR2DS2 and KARAP/DAP12 [3840] . The role of
these receptors on cytolytic CD4 + Tcells, however, is unknown.
In CD8 + cells, NKG2D, for example, can act as a costimulatory
receptor; signaling through NKG2D on these cells can fine-tune
CTL activity in the absence of other costimulatory molecules like
CD28 [4143] . In cytolytic CD4 + Tcells lacking CD28, it has been
hypothesized that NKG2D may serve a similar role [44] .
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Cytolytic CD4 + Tcells in viral immunity

Similarly puzzling is the close phenotypic relationship of cytolytic CD4 + Tcells with Tregs. Tregs have been defined on the
basis of the expression of the transcription factor FoxP3 and
the expression of CD25. While the inhibitory action of Tregs
is unknown, studies of both mice and human cells suggest that
a subset of CD25 + CD4 + T cells can exert cytolytic activity.
CD25 + CD4 + Tcells isolated from human subjects have been
shown to express high levels of perforin and granzyme [45] . Both
iTregs, as well as stimulated CD25 + natural Tregs, were confirmed
to perform MHC-unrestricted killing of autologous targets [46] .
In mice, however, Tregs were observed to lyse targetcells in an
antigen-dependent fashion [47] . Although further studies must
be performed to understand the species and context-specific differences behind these activities, work so far has underscored the
possibility that Treg-mediated cytotoxicity may play an important
role in immune regulation. Most recently, it was demonstrated
that a tumor antigen-specific CD25 +CD4 + cell line was able to kill
dendritic cells when infused into tumor-bearing mice, suggesting
that cytolytic Tregs may represent a mechanism to blunt T-cell
priming by antigen-presenting cells [48] .
Cytolytic effector function

Cytolytic CD4 + Tcells share common cytolytic pathways with

CD8 + CTLs and NK cells. These cell types can kill targetcells
by one of two primary effector mechanisms. In the first, ligation of the Fas ligand (FasL; CD95L/CD178) on effector cells
with its cognate receptor, Fas (CD95), on target cells results in
the activation of caspase-mediated apoptosis programs in the
target[49] . In the second, cytotoxic granules that contain toxic
effector molecules like perforin and granzymes, are released
during exocytotic degranulation, triggering target cell death
both directly and indirectly, by activating downstream apoptosis
pathways [50] .
The earliest mechanistic studies of cytolytic CD4 + Tcells implicated a Fas/FasL-based pathway. Analysis of cytolytic CD4 + T-cell
lines revealed that they killed in a manner that was dependent on
the level of Fas in the target; indeed, Fas-deficient targets from
lpr mice could not be lysed [51] . A similar dependence on the Fas
killing pathway was found in an early invivo study of CD8 + T-celldeficient mice infected with LCMV [52] . However, more recent
reports have also suggested a second, cytotoxic granule-based
mechanism. CD4 + Tcells expressing high levels of perforin and
granzyme or granulysin have been observed in the context of several viral infections as well as in healthy subjects. These cells have
been shown to degranulate upon antigenic stimulation directly
exvivo [27,5355] . A distinct subset of CD4 + Tcells expressing both
perforin and granzymes has been demonstrated in cytomega
lovirus (CMV)-infected patients; these cells can release their
cytotoxic granules in an antigen-specific manner [54] . Likewise,
in HIV-1-infected patients, up to half of HIV-1 Gag-specific CD4 +
Tcells were observed to be able to degranulate, measured by the
expression of CD107a/b (LAMP) on the cell surface [27] .
Studies using perforin inhibitors or cells from perforin-deficient
mice have verified the important role that an exocytotic granule
mechanism plays in CD4 + cytolysis. Treatment of CD4 + T-cell
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clones as well as CD4 + Tcells directly exvivo with concana

mycinA, a vacuolar type H+ -ATPase inhibitor that inhibits exocytotic granule activity, results in a greatly diminished capacity
to lyse autologous targets [36,45] . Although this pathway occurs
by a single general mechanism, it is likely that it can be differ
entially regulated within CD4 + Tcells at the molecular level.
For example, mycobacterium- and EBV-specific cytolytic CD4 +
Tcells in particular have been shown to express high levels of
granulysin [56,57] . In studies of cytolytic Tregs, it was found that
iTregs expressed high levels of granzyme B, while natural Tregs
express granzyme A [46] . The exact conditions that lead to these
differences remain unknown but will probably be important for
vaccine design.
Additional cytotoxic mechanisms have been described, but
their role in mediating cell death is less well studied. In CD8 +
Tcells, it has been demonstrated that the TNF-related apoptosis-inducing ligand (TRAIL) plays a significant role in lysing virally infected targetcells, including in HIV-1 and CMV
infection [58,59] . The function of TRAIL in killing by cytolytic
CD4 + Tcells has been evaluated in a limited number of studies,
which found that CD4 + T-cell clones expressed TRAIL and could
induce bystander apoptosis in antigen-presenting cells [60] as well
as in TRAILsensitive tumor cell lines [61] . However, the role of
the TRAIL pathway in the control of viral infection by CD4 +
Tcells has not been evaluated. Targetcells can be sensitized
for TRAILmediated apoptosis by the presence of inflammatory
cytokines such as TNF-a and IFN-g [59] . In addition to their
ability to potentiate apoptosis by other means, both may also
exert a direct, contact-independent cytotoxic mechanism[62,63] .
It has been shown that both cytokines can lead to cell death
by inducing the production of nitric oxide and other free radicals or by activating death pathways within the targetcell[64,65] .
While cytolytic CD4 + Tcells have been found to secrete these
cytokines, their direct impact on CD4-mediated cytolysis has
not been investigated.
Early efforts to quantify the contributions of the different killing pathways to the overall cytolytic activity of CD4 + Tcells led
to the estimate that Fas/FasL mechanisms accounted for 30% of
killing and the perforin/granzyme pathway played only a negligible role. Cytotoxic cytokines were hypothesized to account
for the remainder of the observed activity [66,67] . However, this
work was performed in a mouse model of graft-versus-host disease and is probably specific for that context. It has also been
proposed that a perforin-based pathway is dominant in humans
and that Fas-mediated killing is the primary cytolytic pathway
for CD4 + Tcells in mice [68] . Evidence in support of this idea,
however, is limited to studies of FasL-deficient mice or patients
showing a hereditary perforin or Fas deficiency. Indeed, more
recent studies in mice have shown that cytolytic CD4 + Tcells
preferentially use a perforin pathway depending on the availability of IL2 [69,70] . Although the impact of killing by CD4 +
Tcells through cytotoxic cytokine secretion or a TRAILbased
mechanism has not been evaluated, it is likely that multiple
pathways coexist and can be differentially induced depending
on the immunological context.
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Differentiation

No specific transcription factor or underlying gene pathway

has been implicated in mediating cytotoxicity of CD4 + Tcells
(Figure1) . Since cytolytic CD4 + Tcells have been suggested to
resemble Th1 cells, a role for the transcription factor Tbet can
be considered. Interestingly, Tbet has also recently been found to
regulate the induction of cytolytic programming in CD8 + Tcells
in combination with another T-box transcription factor, eomesodermin (Eomes) [71,72] , potentially indicating an overlap in the
molecular pathways responsible for cytolytic function in both
cell types.Indeed, transcriptional profiling studies have revealed
remarkable similarities between the transcriptional profiles of
CD4 + and CD8 + Tcells.Both express high levels of perforin,
granzyme and granulysin, among other proteins [73] . Although
the role of Eomes in CD4 + Tcells is not well characterized, it
has been implicated in helping to drive the production of IFN-g
in Th1 cells [11,74] and has been shown to be able to induce the
expression of cytolytic genes when overexpressed in Th2 cells [75] .
Nonetheless, CD4 + effectors have been found to express Eomes at
a considerably lower level than CD8 + CTLs[76] .A related transcription factor, Runt-related transcription factor 3 (Runx3), has
been shown to be important for the induction of Eomes and the
expression of perforin and granzymeB in CD8 + Tcells [77] . In
CD4 + Tcells, Runx3 is known to play a role in Th1 polarization
and IFN-g production[78,79] ; however, whether it is also involved

T regulatory cell
CD25
FoxP3
IL-2
Naive CD4 cell
TGF-

Cytolytic CD4 cell

Unknown
CD27transcription CD28
+/factor
CD57+
NKG2D+

IL-2
IFN-
IL-2
IL-12

Perforin
Granzyme
IFN-

in the induction of cytolytic activity in CD4 + Tcells has not yet

been established. Further transcriptional studies of specific CD4 +
subsets are needed to more rigorously dissect the differences that
lead to cytolytic effector functions in certain CD4 + Tcells.
Recent work by Brown et al. has provided insight into some of the
signals involved in the induction and regulation of cytolytic CD4 +
Tcells in the mouse model [69,80] . Unpolarized CD4 + Tcells as well
as invitro-generated Th1, Th2 and Th17 cells all demonstrated
a cytolytic phenotype, although at significantly varying levels.
While all groups exhibited some degree of cytotoxicity, unpolarized effectors generated in the presence of IL2 alone showed the
greatest cytolytic ability, followed (in order) by Th1, Th2 and Th17
cells[69,80] . The blockade of IL6, IL10, IL12, TNF-a or TGF-b
did not have an effect on the polarization of cells to a cytolytic
phenotype. However, IL2 was necessary for the granule-mediated
killing of peptide-pulsed B-cell targets, while Fas/FasL killing did
not show IL2 dependency [69] . The authors speculated that this
was due to an induction of the perforin/granzyme pathway by
IL2. Indeed, IL2 has a regulatory effect on perforin and granzyme
expression in CD8 + CTLs and the case may be similar for CD4 +
Tcells [81,82] . A role in the induction of cytolytic activity in CD4 +
Tcells has also been proposed for IL15 [36] . Similarly to IL2,
other g-chain receptor cytokines such as IL15 and IL21 can induce
expression of perforin and other cytolytic effector molecules[8385] .
IL15 signaling additionally elicits the preferential production of
CD4 + effector memory cells[86] . Therefore,
while a cytolytic CD4 + T-cell phenotype
can be induced in the presence of high levels of IL2, it is currently unknown whether
the corresponding transcriptional profile is
significantly different from the Th1 CD4 +
T-cell subset.

IL-2
Tbet

Th1 CD4 cell

Figure1. Cytolytic CD4 + T-cell lineage. Cytolytic CD4 + Tcells can resemble both Th1
cells and regulatory Tcells. However, it is unknown whether a cytolytic phenotype
represents a highly differentiated effector state that can be assumed by different CD4 +
subsets, or if cytolytic CD4 + Tcells represent an entirely unique CD4 + T-cell lineage
instead (dotted line). Although acquisition of cytolytic potential seems to be at least
partially dependent on IL2, the underlying transcriptional pathways have not been
determined. Based on the similarities of cytolytic CD4 + Tcells to regulatory Tcells, Th1
and CD8 + Tcells, it is possible that FoxP3, Tbet, Eomes and Runx3 may play important
roles in inducing this activity. Similarly, it possible that g-chain cytokines such as IL15 and
IL21 may participate in the generation of cytolytic CD4 + T-cell responses.

1456

Cytolytic CD4 + Tcells in

HIV-1infection

HIV-1 infection is characterized by progressive infection and depletion of CD4 +

Tcells. This is especially the case in acute
HIV-1 infection, where up to 60% of all
activated memory CD4 + Tcells are infected
and subsequently depleted from all tissue
compartments [87] . Initial HIV-1 vaccine
efforts aimed to avoid any CD4 + T-cell activation to avoid further generation of viral
targetcells. However, recent evidence has
suggested that the induction of CD4 + T-cell
responses might be particularly important
for successful antiviral vaccines [88] . Most
HIV-1-specific CD4 + T-helper responses
are detectable during acute HIV-1 infection, but appear to gradually decline over
the course of viremia [89] . The majority of
HIV-1-specific CD4 + T-cell responses are
directed against Gag and the persistence of
these responses into the chronic phase of the
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Cytolytic CD4 + Tcells in viral immunity

infection has been repeatedly associated with better control of viral

replication [89,90] . In addition to IFN-g secretion, HIV-1-specific
CD4 + T-cell responses also produce TNF-a and IL2, suggesting
a Th1-dominant antiviral CD4 + T-cell response in the absence of
detectable IL4 or IL13 secretion [89] .
Some of the first HIV-specific CD4 + Tcell clones isolated from
infected individuals were able to kill HIV envelope peptide-pulsed
Bcells [91] , suggesting a direct cytolytic potential on the part of
HIV-1-specific CD4 + Tcells. Similar reports described cytolytic
CD4 + Tcells obtained from healthy individuals who received a
recombinant gp160 subunit vaccine during an early HIV-1 vaccine trial [9295] . However, these and most other studies of HIVspecific cytolytic CD4 + Tcells relied on cell lines or clones that
were expanded under long-term culture conditions invitro; it
is therefore difficult to draw conclusions about the relevance of
cytolytic CD4 + Tcells for the control of HIV-1 replication invivo.
Cytolytic CD4 + T cells have also been proposed to be a
byproduct of chronic viral infection, such as CMV or HIV, where
persistent antigenic stimulation could result in the terminally differentiated phenotype that corresponds to cytolytic activity [96] .
In the case of HIV infection, a population of perforin-positive
CD4 + Tcells can be found in all stages of disease progression; this
subset is largest in patients with chronic, untreated disease and can
represent up to half of the CD4 + Tcells in the blood, although
this percentage is highly patient dependent [36] . Additional ana
lysis of lymphocytes from HIV-1-infected patients stimulated with
Gag peptide bolstered these findings, showing that approximately
50% of Gag-specific CD4 + Tcells are able to degranulate, as
measured by the presence of the surrogate degranulation marker
CD107a [27] . Several studies have demonstrated cytolytic activity of CD4 + Tcells from HIV-1patients directly exvivo in the
killing of peptide-pulsed Bcell targetcells in chromium release
assays[97100] . In addition, in a recent Simian immunodeficiency
virus (SIV) study, exvivo peptide-pulsed and fluorescently-labeled
targetcells disappeared after reinfusion, even when test animals
were depleted of CD8 + Tcells [101] . This suggested a non-CD8 +
T-cell-dependent mechanism of targetcell lysis and led the authors
to speculate that cytolytic CD4 + T cells might be involved.
However, a direct contribution of these cells to the control of
viral replication has not been shown. Thus, it is unknown whether
cytolytic CD4 + Tcells are also able to inhibit HIV-1 replication.
Two recent publications have begun to address the question of
whether cytolytic CD4 + Tcells may play a direct role in the control
of virus in HIV-1-infected macrophages or CD4 + Tcells. Sacha
etal. demonstrated that macaques able to control SIV infection
display strong Gag- and Nef-specific CD4 + Tcell responses[102] .
After long-term culture, these CD4 + T-cell responses were able to
inhibit viral replication in SIV-infected macrophages, but were
unable to recognize and kill infected CD4 + Tcells in an invitro
viral replication assay [102] . By contrast, Zheng et al. demonstrated
that Nef-specific CD4 + T-cell clones derived from human elite controllers were able to suppress viral infection in both macrophages
and CD4 + Tcells[103] . Both studies suggested a direct contribution
of cytolytic CD4+ T-cell responses to the control of viral replication.
The concept that cytolytic CD4 + Tcells might recognize virally
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infected macrophages is interesting and potentially important for

HIV-1 vaccine design. Although it has been shown that HIV-1specific CD8 + Tcells are strong contributors to the control of viral
replication, escape mutations in the targeted epitopes can substantially impair their efficiency to recognize and kill HIV-1-infected
cells [104] . Cytolytic CD4 + Tcells, however, recognize targets via
a classII pathway; infected MHC classII-expressing cells, such as
other CD4 + Tcells or macrophages, could therefore be recognized
by a second mechanism. It has also been shown that macrophages
play a role in the pathogenesis of HIV-1 by acting as long-lived
reservoirs for viral persistence [105] . The combined recognition of
virally infected antigen-presenting cells and CD4 + Tcells by both
cytolytic CD4 + and CD8 + cells may therefore play an important
role in HIV-1 pathogenesis. However, it remains to be seen whether
cytolytic HIV-1-specific CD4 + T-cell responses have any effect on
viral inhibition invivo.
Expert commentary

Compelling evidence suggests an unprecedented and critical role

for cytolytic CD4 + effector responses in the control and clearance of pathogens in mice and humans. Although early studies
have already reported on the presence of cytolytic CD4 + Tcells,
their existence has only recently been widely acknowledged and
investigated in greater detail. Especially in viral infectionswhere
pathogens can replicate unhindered within the confines of a
cellrecognition through the antigen presentation machinery
is pivotal for viral containment. Historically, the effector CD8 +
T-cell response has been recognized to be the major contributor to
the control of chronic viral infection. Virus-specific CD8 + Tcells
recognize targetcells after viral antigen presentation through the
MHC class I complex, and significant associations exist between
the expression of certain HLA class I alleles and the ability to
control the replication of viruses like HCV and HIV-1. Thus, there
is strong evidence that CD8 + Tcells play a major role in the lysis
of infected targetcells and therefore contribute to the control of
viral replication. However, CD8 + Tcells are also often unable to
control viral replication alone, emphasizing the need for potential
alternative pathways that can be induced by vaccines.
Although the overall role of cytolytic CD4+ Tcells in the containment of infections invivo remains to be determined, the presence of
these responses raises important considerations for vaccine design.
While MHC class I is ubiquitously expressed, MHC class II expression is limited to professional antigen-presenting cells such as Bcells,
monocytes/macrophages, dendritic cells and some epithelial cells
[106] . The induction of cytolytic CD4 + T-cell responses by a vaccine
might be especially important in the case of HIV-1 infection, where
error-prone reverse transcription during viral replication results in
a vast diversity of circulating strains and the rapid generation of
escape mutations in CD8+ T-cell-targeted epitopes. These escape
mutations often impair CD8 + T-cell recognition and contribute to
an accelerated disease progression [104,107] . Viral immune escape
has also been observed at CD4 + T-cell-targeted epitopes in several
infections, including HCV and the LCMV mouse model [108,109] .
While this phenomenon has not been widely studied to date, CD4 +
T-cell-driven escape mutations have also been suggested to occur in
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Soghoian & Streeck

be particularly important targets of such a

strategy, as these cells are believed to act as
long-lived viral reservoirs [105] .
Eliciting cytolytic CD4 + responses invivo
will require the development of innovative
FasL
vaccines consisting of intelligently designed
Virally infected
Fas
antigens and immunogens. Vaccines designed
antigen-presenting cells
to specifically induce cytolytic CD4 + T-cell
MHC class II
responses have not been studied, primarily
presentation pathway
due to the lack of understanding regarding
their induction. However, efforts to broadly
MHC class II
stimulate CD4 + T-cell responses in general
have been more thoroughly investigated.
HIV
Adenovirus vectors, for example, have been
Perforin
shown to induce potent memory CD4 +
Granzyme A
Granzyme B
T-cell responses [112] . Likewise, vaccination
TNF-
of rhesus macaques with a DNA-prime, adeIFN-
MHC class I
novirus-boost SIV vaccine regimen resulted
presentation pathway
in the generation of durable tissue-wide
virus-specific CD4 + T-cell responses [113] .
MHC class I
In the context of EBV, it was found that
dendritic cells vaccinated with a modified
vaccinia virus Ankara vector could effectively induce both CD4 + and CD8+ T-cell
responses to antigen invitro[114] . DNA vaccination efforts have also been successful at
stimulating CD4 + T-cell responses. A test of
an experimental anti-HIV-1 DNA vaccine
Cytolytic CD8 T cell
in mice revealed that the promiscuous class
II-targeting epitopes encoded by the vaccine
could induce broad CD4+ T-cell responses in
Figure2. Hypothetical model for dual-pathway killing by cytolytic CD4 + and
the context of several different MHC class
CD8 + Tcells. Certain viruses like HIV-1 are able to establish infection within cells that
II molecules [115] . Although the molecular
express both MHC class I and class II molecules, such as antigen-presenting cells. Antigen
mechanisms of antigen processing that lead
presentation by these molecules is critical for recognition by circulating cytolytic CD4 +
to efficient presentation of peptides on MHC
and CD8 + Tcells. Even if the virus were to inhibit recognition through one pathwayfor
instance by downregulating MHC class I molecules or via the acquisition of escape
class II molecules are still unclear, it has also
mutationseffective recognition and lysis of the infected cell could still be achieved
been found that CD4 + responses to specific
through the other pathway.
DNA vaccine epitopes can be strengthened
the case of HIV infection, indicating the potential importance of through the addition of lysosomal targeting sequences [116] . Lastly,
CD4 + T-cell-mediated immune selection pressure in the control of rationally designed adjuvants are likely to be important for the effiviral replication [110] . Moreover, the HIV-1 accessory protein Nef cient targeting of cytolytic CD4 + T-cell responses. Adjuvants like
has the ability to downmodulate the expression of MHC classI Toll-like receptor ligands, cytokines and costimulatory molecules
and therefore facilitates the evasion of CD8 + T-cell recognition can provide the proinflammatory and conditioning signals necesdirectly [111] . Thus, it is possible that the simultaneous induction sary to drive T-cell differentiation towards a particular lineage[117] .
of cytolytic CD4 + T-cell responses as well as CD8 + T-cell responses As new immunogens are designed and as the pathways that lead
bears several advantages in the context of viral infection: first, dual to the generation of cytolytic CD4 + Tcells are uncovered, the sperecognition through MHC class I and II increases the chance of cific stimulation of these responses invivo is likely to move closer
killing virally infected targetcells (Figure2) . Second, the nature of to reality.
the MHC class II structure allows for a greater diversity in the type
Nonetheless, the induction of cytolytic CD4 + T-cell responses
and number of viral epitopes that can be presented in comparison may be a double-edged sword. Research in LCMV-infected mice
to MHC class I. Thus, the potential ability of a vaccine to induce has shown that an induction of CD4 + effector responses can result
virus-specific cytolytic CD4 + Tcells to recognize and kill virally in an increase in inflammation and a corresponding enhancement
infected targets in concert with cytolytic CD8+ Tcells represents of viral pathogenesis [118] . Furthermore, while cytolytic and other
a unique opportunity to contain viral replication early after HIV-1 desirable CD4 + effectors may be stimulated, unwanted regulatory
acquisition. Infected antigen-presenting cells like macrophages may CD4 + responses can also be amplified, potentially dampening any

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Cytolytic CD4 T cell

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Cytolytic CD4 + Tcells in viral immunity

therapeutic response. Possible virus-specific pitfalls must also be

considered. Activation and generation of virus-specific cytolytic
CD4 + T-cell responses by a preventative vaccine may put patients at
greater risk for HIV-1 infection, for example, as activated memory
CD4 + Tcells have been shown to be preferentially targeted by
the virus [10] . In EBV infection, where cytolytic CD4 + Tcells are
recognized to play an important role in viral suppression, it has also
been found that EBV-specific CD4 + T-cell responses can reactivate
latently infected cells, possibly leading to the proliferation of cancerous transformed B cells [119] . More broadly, the function and role
of cytolytic CD4 + Tcells invivo must be evaluated to determine
their impact on the modulation of other antigen-specific B and
CD8 + T-cell responses.
Fiveyear view

Within the next 5 years, considerable advances in our under

standing of cytolytic CD4 + T cells are expected. One specific
unanswered question is precisely when CD4 + Tcells commit to the
cytolytic CD4 + T-cell program during their lineage development.
It is currently unknown if cytolytic activity is simply a function
acquired by terminally differentiated CD4 + Th1 cells or Tregs,
or if it represents a characteristic of a unique CD4 + T-cell subset
(Figure1) . We expect that careful multiparametric analysis of the
various CD4 + T-cell phenotypes that have shown cytolytic potential, in combination with detailed microarray profiling, will shed
further light on this question. The identification of specific markers
or transcriptional profiles will lead to a better exvivo assessment of

Review

cytolytic CD4 + T-cell responses and therefore help to evaluate their

role in different disease settings. A closely related question and
one especially relevant in the context of vaccine design is how can
cytolytic CD4 + responses be induced and/or maintained invivo?
Here it will be important to understand which antigens but also
whether specific adjuvants play a role in the directed stimulation
of cytolytic CD4 + T-cell responses. Vaccine trials in animals will
start to elucidate if the induction of such responses can bring about
better results than the primary induction of antigen-specific cytolytic CD8+ Tcells. In terms of HIV-1 vaccine design, we will obtain
a better understanding of whether the induction of cytolytic CD4 +
Tcells is beneficial for viral control or rather leads to the generation
of activated targetcells and accelerates disease progression. Finally,
in the next few years, we expect to develop a better understanding of the differences between cytolytic CD4 + and CD8 + T-cell
responses and under which circumstances one or the other pathway
is preferentially elicited. This knowledge will be pivotal in guiding
rational, reverse vaccine design, especially against viral infections
that have so far eluded efforts to create effective vaccines.
Financial & competing interests disclosure

Hendrik Streeck is funded by NIH grant 1R01AI091450-01 and the Bill

and Melinda Gates Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a
financial interest in or financial conflict with the subject matter or materials
discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Key issues
Cytolytic CD4 + Tcells have been shown to play a role in numerous viral infections, both invitro and invivo. These cells can directly kill
infected targetcells in an MHC class II-restricted manner.
Cytolytic CD4 + Tcells can kill infected targets by several pathways, including Fas/Fas ligand or granule-based mechanisms.
Although the mechanisms that lead to the induction of cytolytic CD4 + responses remain unknown, several subsets of CD4 + Tcells
havebeen shown to exhibit cytolytic potential, including Th1 cells and Tregs. This potential is often correlated with markers of
terminaldifferentiation.
Induction of cytolytic CD4 + Tcells by vaccines may represent an important mechanism by which the activity of CD8 + Tcells could be
complemented to inhibit viral replication.
Future research should focus on characterizing the importance of cytolytic CD4 + T-cell responses invivo, as well as establishing how
such responses could be induced though vaccine efforts.
lymphocytes by a soluble factor released
from antigen-stimulated T lymphocytes.
J.Exp. Med. 138(5), 12301247 (1973).

One of the first reports of cytolytic Tcells

that are able to recognize antigen
presented on MHC class II.

Reinherz EL, Schlossman SF. The

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Reinherz EL, Kung PC, Goldstein G,

Schlossman SF. Separation of functional
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Meuer SC, Schlossman SF, Reinherz EL.

Clonal analysis of human cytotoxic T
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Krensky AM, Reiss CS, Mier JW,

Strominger JL, Burakoff SJ. Long-term
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