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NK cells controlling virus-specific T cells: Rheostats for acute vs. persistent infections

Department of Pathology and Program for Immunology and Virology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA. Electronic address: .
Virology (Impact Factor: 3.32). 01/2013; 435(1):37-45. DOI: 10.1016/j.virol.2012.10.005
Source: PubMed
ABSTRACT
Viral infections characteristically induce a cytokine-driven activated natural killer (NK) cell response that precedes an antigen-driven T cell response. These NK cells can restrain some but not all viral infections by attacking virus-infected cells and can thereby provide time for an effective T cell response to mobilize. Recent studies have revealed an additional immunoregulatory role for the NK cells, where they inhibit the size and functionality of the T cell response, regardless of whether the viruses are themselves sensitive to NK cells. This subsequent change in T cell dynamics can alter patterns of immunopathology and persistence and implicates NK cells as rheostat-like regulators of persistent infections.

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Available from: Stephen N Waggoner
Review
NK cells controlling virus-specific T cells: Rheostats for acute vs.
persistent infections
Raymond M. Welsh
n
, Stephen N. Waggoner
Department of Pathology and Program for Immunology and Virology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA
article info
Keywords:
Natural killer (NK) cells
T cells
NK cell receptors (NKR)
2B4
Lymphocytic choriomeningitis
virus (LCMV)
Murine cytomegalovirus (MCMV)
Persistent infection
Pathogenesis
Interferon
Cytotoxic
abstract
Viral infections characteristically induce a cytokine-driven activated natural killer (NK) cell response
that precedes an antigen-driven T cell response. These NK cells can restrain some but not all viral
infections by attacking virus-infected cells and can thereby provide time for an effective T cell response
to mobilize. Recent studies have revealed an additional immunoregulatory role for the NK cells, where
they inhibit the size and functionality of the T cell response, regardless of whether the viruses are
themselves sensitive to NK cells. This subsequent change in T cell dynamics can alter patterns of
immunopathology and persistence and implicates NK cells as rheostat-like regulators of persistent
infections.
& 2012 Elsevier Inc. All rights reserved.
Contents
Introduction. . .........................................................................................................37
Patterns of viral pathogenesis and persistence . ..............................................................................38
What we know about how NK cells control viral infections . ....................................................................39
Influence of NK cells on T cell responses ....................................................................................40
NK cells regulate viral pathogenesis of an NK-‘‘resistant’’ virus by regulating the T cell response .......................................40
NK cell lysis of CD4 T cells is a general feature of viral infections ................................................................42
NKR involved in the NK cell regulation of T cell responses. . ....................................................................42
Genetic studies linking NKR to persistent infecti ons in humans..................................................................42
Influence of NK cells on the generation of T cell memory. . . ....................................................................43
Conclusions . . .........................................................................................................43
Acknowledgments ......................................................................................................43
References . . . .........................................................................................................43
Introduction
Natural killer (NK) cells and T cells are regulatory and effector
lymphocytes that get mobilized into the host response to viral
infections. NK cells are cytotoxic antiviral cytokine-producing
lymphocytes whose activities are regulated by cytokines and by
a number of stochastically-expressed positive- and negative-
signaling NK receptors (NKR) that recognize cellular stress-
related molecules, adhesion molecules, and major histocompat-
ibility complex (MHC) proteins (Lanier, 2008; Raulet, 2003). Some
NKR have even evolved to directly recognize certain viral proteins
(Daniels et al., 2001; Lee et al., 2001; Brown et al., 2001; Voigt
et al., 2003). T cells, on the other hand, express randomly
generated and clonally distributed T cell receptors (TCR) that
recognize processed viral peptide epitopes presented to them in
the grooves of MHC molecules expressed on the surface of
antigen-presenting cells (Wilson et al., 2004). The activated T
cells can be similar to NK cells in their acquisition of cytotoxic and
Contents lists available at SciVerse ScienceDirect
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Virology
0042-6822/$ - see front matter & 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.virol.2012.10.005
n
Corresponding author. Fax: þ1 508 856 0019.
E-mail address: Raymond.Welsh@umassmed.edu (R.M. Welsh).
Virology 435 (2013) 37–45
Page 1
cytokine-producing effector functions; this is especially true for
the CD8 T cells, which recognize peptide epitopes presented by
class 1 MHC molecules. The activated CD4 T cells, which recog-
nize peptides presented by class 2 MHC molecules, can secrete
factors that regulate the T cells and the rest of the immune
response in positive or negative ways. NK cells patrol the host at a
moderate state of activation and at a relatively high frequency
( 15% of peripheral blood lymphocytes), but will proliferate and
become even more active during a viral infection (Biron et al.,
1983; Welsh, 1978). However, immunologically naı
¨ve
T cells
specific to any peptide epitope exist at low frequency
( 1/50,000) and in an inactive naı
¨ve
state and require a sub-
stantial clonal expansion to increase in numbers and functions
sufficient to control of infection (Blattman et al., 2002; Seedhom
et al., 2009). Innate cytokines such as the type 1 interferons (IFN),
IL-12, and IL-15 are rapidly induced during viral infections and
can stimulate the activation and proliferation of NK cells and
greatly augment the proliferation of T cells (Biron, 1995). The
dynamics of this process follow the innate and adaptive immune
response paradigm, first described in the 1970s: an early
cytokine-driven activated NK cell innate response followed by a
peak in clonally expanded T cells (Fig. 1)(Welsh, 1978).
The temporal relationship between the early activated NK cell
vs. late T cell peak has historically engendered questions about
whether these cell populations were influencing each other.
Certainly, the T cell response may clear the pathogen that induces
the cytokines that the NK cells need to stay highly active and
proliferating. That is probably not the entire explanation of the
waning of the NK cell response, however, as some work has
shown that TGF
b
made late in the response has a more suppres-
sive effect on NK cells than T cells (Su et al., 1991). In the other
direction, a number of papers described later have proposed that
NK cells may either enhance or inhibit the T cell response, and
earlier papers even suggested that the NK cells may turn into
T cells! We can dismiss that latter suggestion, as it is now clear
that NK cells and T cells represent different lineages, but the
question of how well NK cells control T cells has recently come to
the forefront. It would not be out of the question to think that NK
cells could promote T cell proliferation, as they produce IFN
g
,
which itself can promote CD8 T cell expansion (Whitmire et al.,
2007). Also, NK cells might indirectly promote T cell expansion by
directly controlling viral load early in infection, thereby inhibiting
the levels of virus that might cause immune suppression
(Bukowski et al., 1984). It should also be of no surprise that NK
cells could affect T cells in a negative way. T cell targets such as
mouse YAC-1 cells were among the earliest target cells used in
cytotoxicity assays to detect the activity of NK cells (Salazar-
Onfray et al., 1997), and primary thymocytes were among the first
documented targets in vivo (Hansson et al., 1980, 1979. Recent
work has indicated that in the context of a viral infection the NK
cells have the capacity to directly kill or indirectly regulate the
numbers and activities of antiviral CD4 and CD8 T cells (Su et al.,
2001; Waggoner et al., 2010, 2012; Lang et al., 2012; Narni-
Mancinelli et al., 2012; Andrews et al., 2010; Robbins et al., 2007;
Mitrovic et al., 2012; Ge et al., 2012; Lee et al., 2009; Stadnisky
et al., 2011). As a consequence of this activity, NK cells may serve
as rheostats regulating the T cells that control whether an
infection becomes resolved, persistent, or lethal.
Patterns of viral pathogenesis and persistence
Viral infections can present themselves in many forms. Many
acute viral infections induce sterilizing T and B cell immune
responses that clear the infection, form long term memory, and
leave the host resistant to re-infection. Other infections, such as
NK Cell
Activity
αβT Cell
Activity
Innate
Cytokines
Fig. 1. Innate and adaptive host response to infection. This figure portrays the timing of the peaks in innate cytokines (type 1 IFN, etc.), NK cell cytolytic activity (not cell
number), and T cell number and activity during an acute viral infection, based on Welsh (1978).
R.M. Welsh, S.N. Waggoner / Virology 435 (2013) 37–4538
Page 2
those with a variety of herpesviruses, are mostly cleared by the
immune response, but residual foci of low level infection remain,
with the possibility of an occasional reactivation. Other viruses
can cause long term persistent infections associated with high
levels of virus due to some compromise of immunological func-
tion. Examples of these abound, including hepatitis B and C and
HIV in humans and lymphocytic choriomeningitis virus (LCMV) in
the mouse. The balance between the functional immune response
and the viral load is crucial in determining the pattern of viral
persistence. An overzealous immune response in the presence of
high antigen load can lead to severe immune pathology such as
that which occurs during fulminant viral hepatitis. This means
that in some cases it might be good to generate a very strong
immune response to quickly eradicate viral antigen, but in other
cases, in order to protect the host from severe immune pathology,
a more tempered response may be better.
This paradox can perhaps best be illustrated in mice intrave-
nously infected with the clone 13 strain of LCMV (Waggoner et al.,
2012). This strain, in part due to its high affinity binding to its
alpha dystroglycan receptor on host cells (Cao et al., 1998),
disseminates rapidly in adult mice and can cause a persistent
infection when inoculated at high dose (Ahmed et al., 1984). This
persistent infection can occur because the virus is not directly
cytopathic and because the functions of the T cells that would
normally control the infection become compromised. The T cell
response becomes clonally exhausted, in part by the activation-
induced death of high affinity T cells (Zhou et al., 2002) and in
part by inhibitory T cell signaling molecules that provide negative
signals to T cells challenged with high antigen load (Zajac et al.,
1998; Barber et al., 2006). These include molecules such as
programmed death-1 (PD-1/CD279), 2B4 (CD244), lymphocyte
activation gene-3 (LAG-3/CD233), CD160, and cytotoxic T lym-
phocyte antigen 4 (CTLA-4) (Wherry, 2011). At low virus dose a
rapid and fully functional T cell response clears LCMV clone 13
with little immune pathology (Fig. 2). However, at an intermedi-
ate dose of virus there is a high enough antigen load and a
sufficiently functional T cell response that severe immune patho-
logy develops in the liver and lungs (Waggoner et al., 2012;
Stamm et al., 2012). Thus the magnitude and functionality of a T
cell response will determine whether a viral infection results in
clearance, persistence, or sometimes lethal immune pathology.
The host has mechanisms to regulate such responses, and new
findings indicate that NK cells can act like rheostats to monitor
anti-viral T cell responses. In the LCMV model described above,
depletion of NK cells prevents the severe pathology in the mid-
dose model and leads to viral clearance; here we can say that the
presence of NK cells was bad for the host (Fig. 2). On the other
hand, depletion of NK cells in the high dose model results in high
mortality (Waggoner et al., 2012). There the NK cells are appar-
ently needed for the persistent infection to develop. Otherwise
the T cell response is so severe that lethal immune pathology
occurs.
What we know about how NK cells control viral infections
NK cells are thought to directly control infections with
some viruses if the NK cells become positively activated by the
up-regulation of stress-related proteins in virus-infected cells or
if they fail to respond to negative signals that may occur, for
example, by the down regulation of class 1 MHC antigens on
virus-infected cells (Raulet, 2003; Brutkiewicz and Welsh, 1995).
As a consequence, viruses may encode proteins to interact with
these negative and positive signaling triggering molecules or their
ligands to alter the dynamics of this process. This sometimes can
be tricky, as T cells require MHC molecules for recognition of
targets. Thus, protecting targets from T cells by the down
modulation of MHC antigens might make those targets more
sensitive to NK cells. Type 1 IFN, which is induced at high levels
during viral infection, up-regulates expression of class 1 MHC, and
in so doing increases target cell sensitivity to CTL while decreas-
ing sensitivity to NK cells (Trinchieri and Santoli, 1978; Welsh
et al., 1981;
Ljunggren and Karre, 1990; Bukowski and Welsh,
1986). Interestingly, HIV nef protein down regulates the HLA- A
and B MHC molecules, with which CD8 T cells prefer to react,
while allowing cell surface expression of HLA-C, with which NK
cells prefer to react (Cohen et al., 1999; Swann et al., 2001). When
NK cells do directly control viral infections they do so by way of
perforin-dependent cytotoxicity or by the secretion of IFN
g
,
which has many downstream consequences, including the induc-
tion of nitric oxide synthase, whose metabolite, nitric oxide, has
dramatic anti-microbial properties. With murine cytomegalovirus
(MCMV) for which the most is known about the NK cell-mediated
control of viral infections, a perforin-dependent mechanism is
preferred in the spleen, and an IFN
g
- and NO-dependent mechan-
ism is preferred in the liver (Tay and Welsh, 1997).
The MCMV infection of the C576BL/6 mouse is the best studied
model for the control of viral infections by NK cells. The genetics
of resistance maps to the NK cell complex on chromosome 6
(Scalzo et al., 1995). This gene complex codes for a series of
proteins of interest to NK cells, including the positive signaling
molecules NKR-P1c (NK1.1), a target for a useful depleting anti-
body, and NKG2D. It also codes for CD94 and NKG2A, B, C, and E
proteins, which interact to form negatively signaling heterodi-
mers, and a group of c-type lectin molecules of the Ly49 series,
which deliver negative or positive signals when engaging discrete
mouse MHC molecules (Brown et al., 1997). The NKG2 and CD94
molecules have human homologs, as does the positively signaling
receptor NKp46, which is encoded on chromosome 7 (Biassoni
et al., 1999). The Ly49 molecules do not have human homologs,
but human killer cell immunoglobulin-like receptors (KIRs) per-
form the same function of recognizing MHC molecules (Lanier,
1998). What is unusual in the C57BL/6 mice is that the genetic
resistance to MCMV maps to a Cmv-1 resistance locus that is
congruent with Ly49H, a positive signaling NKR that reacts with
the MCMV-encoded glycoprotein m157, thereby initiating an NK
cell attack on MCMV-infected cells (Brown et al., 2001; Smith
et al., 2002; Daniels et al., 2001; Lee et al., 2001; Arase et al.,
2002). Passage of MCMV in Ly49H-expressing mice selects for
m157 negative viral mutants (Voigt et al., 2003). m157, however,
is just one of a family of similar molecules encoded by MCMV, and
other molecules of this family interact with other Ly49 molecules,
Fig. 2. Pathogenesis of LCMV infection at different viral doses in normal or NK
cell-depleted mice. The red area is the zone of severe immune pathology, where
there are sufficient amounts of virus and T cells to cause tissue damage. At a
higher dose of virus the T cells exhaust, and at a lower dose the virus is cleared. NK
cell depletion results in a stronger T cell response, which changes the dose at
which immune pathology occurs (Waggoner et al., 2012).
R.M. Welsh, S.N. Waggoner / Virology 435 (2013) 37–45 39
Page 3
such that the genetics of resistance to MCMV differs between
mouse strains (Kielczewska et al., 2009; Orr and Lanier, 2011;
Lanier, 2008). MCMV also encodes proteins that alter the expres-
sion of cellular class I proteins, presumably to confuse both NK
cells and T cells (Babic et al., 2010). Further, MCMV encodes
proteins that inhibit expression of the ligands for NKG2D
(Arapovic et al., 2009). The complexity of the MCMV system
may be due to the fact that MCMV causes a persistent infection
and has co-evolved with its host for millions of years. Human
CMV is similar in regards to its complex relationship with its host
(Lanier, 2008).
Less is known about the NK cell recognition of other viruses.
Genetic resistance to ectromelia virus also maps to the NK
complex, and this resistance seems in part to be mediated by
NKG2D (Fang et al., 2008) and by CD94-NKG2E heterodimers
(Fang et al., 2011). Resistance to influenza A virus (IAV) has been
attributed in part to NKp46, as this molecule is reported to
interact with the IAV hemagglutinin (Glasner et al., 2012), and
NKp46 knock-out mice have enhanced sensitivity to IAV (Gazit
et al., 2006). NK cells do not seem to directly control mouse
polyomavirus, but NK cells lyse polyomavirus-induced tumors by
an NKG2D-dependent mechanism, and tumor growth is enhanced
in NK cell-deficient mice (Mishra et al., 2010).
Other viruses resist the direct anti-viral effects of NK cells. An
example is LCMV, which grows to equal titers during the first
three days of infection in the spleens of mice depleted or not of
NK cells, and which grows similarly in SCID mice deleted or not of
NK cells for many weeks (Welsh et al., 1991). The relatively
noncytopathic LCMV infection does not cause appreciable stress
to cells nor does it down-regulate class I MHC antigens (Bukowski
and Welsh, 1985). Although IFN
g
production by NK cells may
slightly restrict LCMV replication at the peripheral inoculation
site (Mack et al., 2011), we can generally categorize LCMV as a
relatively NK-resistant virus and contrast it to MCMV, the proto-
typic NK-sensitive virus. Nevertheless, depletion of NK cells under
certain conditions can greatly alter the patterns of LCMV patho-
genesis and persistence (Fig. 2). This effect of NK cells is not,
however, because of any direct effect of NK cells on LCMV. Rather,
it reflects the ability of the NK cells to modulate the T cell
response.
Influence of NK cells on T cell responses
Some studies in the 1980s using reagents inferior to what we
have now suggested that CTL development may be dependent on
NK cells (Burlington et al., 1984; Suzuki et al., 1985), whereas
other reports had implicated NK cells as being natural suppres-
sors of immunological function, inhibiting hematopoiesis
(Kiessling et al., 1977; Thomsen et al., 1986) and B cell prolifera-
tion and differentiation (Arai et al., 1983; Kuwano et al., 1986).
One of the earliest seminal papers on NK cells by Kiessling et al.
(1977) linked NK cells to the control of bone marrow allografts.
A subpopulation of class 1 MHC low thymocytes were shown to
be highly sensitive to NK cell-mediated lysis (Hansson et al., 1979,
1980), and in vivo generated NK cells were shown to lyse other
NK cells or cell lines mediating NK-like cytotoxic activity. Even
IL-2-stimulated ‘‘lymphokine activated killer (LAK) cells’’, some-
times used in tumor therapy regimens, could be killed by
activated host NK cells when inoculated in vivo (Brubaker et al.,
1991). A number of studies have also focused on the potential
ability of NK cells to lyse dendritic cells (Wilson et al., 1999;
Gilbertson et al., 1986; Andrews et al., 2003), which not only
provide cytokines for the activation of NK cells but also present
class 1 and 2 MHC antigens and stimulate CD8 and CD4 T cell
responses. Thus, the potential is there for NK cells to be effective
modulators of immune response function.
Nevertheless, the literature on whether NK cells can have an
impact on viral pathogenesis by an immunoregulatory mechan-
ism has been murky until recently. Most of the studies had been
done in the MCMV system, where depletion of NK cells causes
elevations in viral titers and virus-induced cytokines simply due
to the removal of the direct NK cell control of the virus. This
obfuscates the interpretation of downstream events after NK cell
depletion. Some reports using such NK cell depletion strategies
have suggested that NK cell depletion enhances MCMV-induced T
cell responses (Su et al., 2001; Andrews et al., 2010) and other
reports suggest that NK cell depletion suppresses MCMV-induced
T cell responses (Bukowski et al., 1984; Robbins et al., 2007).
We feel that a possible explanation for this dichotomy may relate
to the effect of NK cell depletion on viral load in the animal.
A certain threshold of viral antigen and virus-induced cytokines is
needed to induce a strong CD8 T cell response. Thus, if the NK cell
depletion enhances a low virus load to a moderate virus load, an
elevated T cell response may occur. On the other hand, if the NK
cell depletion converts a moderate viral load into a high viral load,
then the viral antigens and high cytokine levels may cause a
general immune suppression and reduced T cell response, as
detailed by us previously (Bukowski et al., 1984). In a recent
elegant though unusual mutagenesis study in mice, hyperactive
NK cells were generated due to a mutation in the Ncr1 gene that
encodes the positively signaling NK cell receptor NKp46. These
mice had enhanced NK cell activity, enhanced resistance to
MCMV, and a reduced CD8 T cell response to MCMV (Narni
-
Mancinelli et al., 2012). Some work with MCMV has indicated that
the activated NK cells may be affecting T cell responses by killing
off the dendritic cells or other antigen-presenting cells (Andrews
et al., 2010). However, in this case the DC would be infected with
MCMV and be susceptible to a virus-specific NK cell attack. The
consequential loss of DC may well alter the T cell response, but it
is difficult to attribute this to a true immunoregulatory role for NK
cells rather than just to their ability to kill virus-infected cells.
Thus, for a less ambiguous read out, studies have recently been
done with a virus, LCMV, that is not directly controlled by NK cells.
NK cells regulate viral pathogenesis of an NK-‘‘resistant’’ virus
by regulating the T cell response
For many years the Armstrong strain of LCMV was routinely
studied by our group in NK-depleted vs. normal mice, and only
modest differences in T cell activity were observed in the
presence vs. the absence of NK cells. Beige mice, which have a
defect in NK cell cytolytic activity, had higher levels of T cells than
wild type mice after LCMV-Armstrong infection, but that could
have been due to a slower clearance of the virus by the T cells,
which were also cytolytically compromised (Biron et al., 1987).
More recently Su et al. found little difference in anti-viral T cell
responses in LCMV-Armstrong infected wild type mice depleted
of NK cells, but, surprisingly, they found that the CD4 T cell
response in
b
2 microglobulin knock-out mice, which have very
few CD8 T cells, was elevated in the absence of NK cells (Su et al.,
2001). Our recent studies have shown that NK cells can drama-
tically regulate the T cell responses in other models or conditions
of LCMV infection and that they do so by cytolytically attacking
the CD4 T cells, consistent with the findings with the
b
2 micro-
globulin knock-out mice (Waggoner et al., 2012).
By exploiting the various pathogenic models of the clone 13
variant of LCMV, we were able to demonstrate profound impacts
of the NK cells on T cell responses and viral pathogenesis, as
mentioned earlier in this review (Waggoner et al., 2012)(Fig. 2).
R.M. Welsh, S.N. Waggoner / Virology 435 (2013) 37–4540
Page 4
At a low virus dose NK cell depletion resulted in a higher CD8
T cell response which had no major role in the already effective
clearance of the virus. At the medium dose, which normally
resulted in severe and often lethal T cell-dependent immune
pathology, the deletion of NK cells prevented the disease. The
reason that the disease was prevented was that the absence of the
NK cells allowed for a much greater CD4 and CD8 T cell response
in terms of virus-specific T cell number and functionality. Many
of the CD4 and CD8 T cells present in medium dose-infected
untreated mice had a partially exhausted phenotype and pro-
duced few cytokines. The reason for the reduction in pathology in
the NK cell-depleted medium-dose infected mice was simply due
to the enhanced clearance of virus by the more vigorous T cell
response. Thus, at this dose the presence of NK cells was
detrimental to the host, because it inhibited a T cell response
required to clear virus before the acquisition of severe immune
pathology. It should be emphasized that all the pathology was
mediated by the T cells. In medium dose-infected T cell knock-out
mice there was no weight loss, no mortality, no significant
immune pathology, and no clearance of virus, and no differen-
ces in viral titers, consistent with the fact that LCMV is an
NK-resistant virus and that the effects of the NK cell depletion
were mediated by T cells.
Paradoxically, depletion of NK cells at a high virus dose that
would normally induce a several month persistent infection
without mortality resulted in enhanced pathology and mortality.
Briefly put, for this well-established model for viral persistence,
NK cells were needed for the persistence to occur. The reason for
the increased mortality in the absence of NK cells appeared to be
due to the fact that the larger and more effective T cell response in
the absence of NK cells could not be properly exhausted before
severe damage was done.
These changes in viral pathogenesis were all accomplished by
a single injection of a limiting amount of NK cell depleting
antibody at the beginning of infection. This regimen depleted
classic NK cells and not NK/T cells. Further, this could be
accomplished with either of two NK cell-depleting antibodies
and similarly occurred in CD1d KO mice which lack many NK-T
cells and in
gd
TCR KO mice, which lack NK1.1-expressing
gd
T
cells (Waggoner et al., 2012). Thus, eliminating NK cells at their
peak of activation had a profound effect. We more recently have
examined whether depletion of NK cells several weeks into a
persistent infection could resurrect T cell responses and stimulate
viral clearance. Our preliminary data indicate that this treatment
can indeed have therapeutic value, and we are testing this further.
We explored the mechanism by which the NK cells controlled
the T cell response in this LCMV model and found, using in vivo
cytotoxicity assays described in Fig. 3, that the NK cells cytolyti-
cally attacked activated CD4 T cells in a perforin-dependent
manner (Waggoner et al., 2012). Further, in the medium dose
model, depletion of NK cells affected CD8 T cell numbers and
function only if the CD4 T cells were present. From these data we
proposed a model whereby the effects of NK cells on CD4 T cells
were mostly direct, due to NK cell-dependent cytotoxicity, but the
effects of NK cells on the CD8 T cells were indirect. Thus, the
complete model, as shown in Fig. 4, indicates that (a) a virus
infection induces cytokines and antigen to activate the NK cells
and T cells, (b) the activated NK cells cytolytically eliminate some
of the activated CD4 T cells, which are needed to provide help for
the (c) CD8 T cells, which control the viral load that can, in turn,
(d) functionally exhaust the T cells if the antigen load gets too
high. The pathological consequences of these events are depen-
dent on a numbers game between the degree of antigen load and
the magnitude of the T cell response, and the rheostat-like effect
the NK cells can have on these dynamics.
A story with some similarities has been reported using a
different persistent infection-inducing strain of LCMV, the
Docile variant of strain WE (Lang et al., 2012). NK cell-deficient
Fig. 3. In vivo cytotoxicity assay. Splenocytes from NK cell-depleted, virus-infected Ly5.1þ mice are labeled with CFSE and transferred into infected or uninfected
recipients deleted or not of NK cells. After 5 h donor target cells are gated for CD4 or CD8, and the numbers of cells expressing activation antigens (e.g. CD43 and CD44) are
quantified (Waggoner et al., 2012).
Fig. 4. NK cells regulate the T cell response by acting on CD4 T cells. This
diagrams how NK cells can regulate the overall T cell response by attacking
activated helper CD4 T cells. This allows for excess viral antigen that can exhaust
both CD4 and CD8 T cells.
R.M. Welsh, S.N. Waggoner / Virology 435 (2013) 37–45 41
Page 5
(Nfil3-/-, E4BP4-/-) mice infected with this virus and wild type
mice depleted of NK cells with antibody developed enhanced T
cell responses to LCMV. Under the dose of virus used, the absence
of NK cells enabled a stronger T cell response that prevented the
persistent infection. This report focused on the effects of NK cell
depletion on CD8 T cell activity and showed that perforin-
containing NK cells could limit the proliferative expansion of
CD8 T cells. This conclusion was based on an indirect assay, and a
role for CD4 T cells was not addressed.
NK cell lysis of CD4 T cells is a general feature of viral
infections
NK cell-mediated regulation of T cell activity may be a general
property of viral infections, but the impact of NK cells on
infections with certain viruses may be complicated by the fact
that NK cells can possess direct anti-viral activity and at the same
time suppress the T cell response. Thus, the positive and negative
effects of the NK cells have the potential to cancel each other out.
This could be a particular problem in the analysis of the MCMV
system, where the virus is clearly sensitive to control by NK cells,
CD4 T cells, and CD8 T cells. The other issue in evaluating what
might be enhanced T cell responses after NK cell depletion is that
a more rapid T cell-dependent clearance of antigen in the absence
of NK cells might reduce the antigen load needed to further
increase the T cell response. This may well be the story with the
Armstrong strain of LCMV, which has a relatively restrained
replication and with which there had not been a noticeable
phenotype found after NK cell depletion. We reinvestigated this
and found that if one looked earlier after infection (day 9), there
was an enhanced CD8 T cell response as well as enhanced
clearance of the virus. By day 98 there was little difference in
the T cell response, as the T cell response in the untreated
animals, which bore a higher early viral load, ‘‘caught up.’’ Thus,
by examining the T cell response at its peak the impact of NK cell
depletion had gone unnoticed.
Given all these issues, we sought to directly examine the
effects of NK cells induced by several viruses on activated CD4 T
cell targets induced by several viruses. By employing the in vivo
cytotoxicity assay (Fig. 3) we could mix and match target and
effector populations and bypass issues presented by the direct NK
cell control of viral infections or changes in antigen clearance
dynamics by the activated T cells. The results showed that NK
cells activated by LCMV, Pichinde virus, mouse hepatitis virus, or
the IFN-inducer poly I:C all could lyse LCMV-induced activated
CD4 T cells in vivo. Further, CD4 T cells activated by infections
with LCMV, MCMV, Pichinde virus, mouse hepatitis virus, or
vaccinia virus were all susceptible to lysis by LCMV-activated
NK cells (Waggoner et al., 2012). This indicates that activated NK
cell-mediated killing of activated CD4 T cells is a universal
phenomenon occurring during viral infections.
NKR involved in the NK cell regulation of T cell responses
In vitro studies have shown that NK cells can kill activated CD8
T cells by way of NKG2D, which recognizes stress-related ligands
on target cells (Rabinovich et al., 2003), and Lang et al. (2012)
have reported that NKG2D is responsible for the NK cell regula-
tion of CD8 T cell responses in their system with the Docile LCMV
strain. They report modest expression of NKG2D ligands on the
CD8 T cells and a higher CD8 T cell response in mice treated with
an anti-NKG2D blocking antibody. In our Clone 13 LCMV system,
which employed the in vivo cytotoxicity assay, there was very
little expression of NKG2D ligands on activated CD4 or CD8
T cells, and the NK cell-dependent killing of the activated CD4
T cells occurred normally in the presence of anti-NKG2D or in
NKG2D KO mice (Waggoner et al., 2012). It is quite possible that
different NKR are used in different infection systems, but this
awaits further clarification.
Regulation of NK cell killing of T cells by negative-signaling
receptors, however, seems more clear to us. 2B4 (CD244) is an
NKR that can deliver negative signals to NK cells after engaging its
ligand, CD48, which is expressed on many cell types (Lee et al.,
2004). In the clone 13 system, where activated CD4 T cells are
more sensitive to lysis than activated CD8 T cells, expression of
CD48 was much higher on the CD8 T cells (Waggoner et al., 2012).
Further, during infection of 2B4 KO mice, the NK cells aggressively
and directly lysed activated CD8 T cells (Waggoner et al., 2010).
Having described how the presence or absence of NK cells can
affect viral persistence, we used 2B4 KO mice to examine how NK
cells without their normal inhibitions would act in the context
of a persistent infection. High dose clone 13 infection of 2B4
KO mice resulted in reduced T cell responses, thymic atrophy,
splenomegaly, chronic hepatitis, and a long term persistent
infection that never resolved (Waggoner et al., 2010). All of these
pathologies were ablated by a single injection of anti-NK1.1 in the
first three days of infection, and the infection ultimately resolved,
like that in wild type mice. Thus, in wild type mice the presence
of
NK cells enable a persistent infection that resolves in a few
months; the absence of NK cells in wild type mice causes an
aggressive immune response and severe immune pathology and
death; and hyperactive NK cells in 2B4 KO mice severely ablate the
T cell response and result in a persistent infection that does not
resolve. Hence, the activity of NK cells very early in the infection
process can have a long lasting impact on viral persistence.
Genetic studies linking NKR to persistent infections in humans
Many human viral infections result in long term persistence, and
HIV, HBV, and HCV are highly problematic infections associated
with high virus loads. All of these infections are thought to be
controlled at least in part by T cells, but why different HIV-infected
individuals have different antigen load set points and why some
hepatitis virus infections resolve and others do not has remained a
mystery. A series of human genetic studies has been performed by
Carrington and co-workers in an attempt to correlate the pathogen-
esis of these infections with the presence of certain NKR, notably
the KIR, and the MHC antigens with which they engage (Carrington
and Alter, 2012). Strikingly, several KIR–MHC combinations have
shown correlations with control of infection, and the question is
why, with the answer probably being quite complex. There appears
to be KIR-mediated selection of HIV variants that might suggest
direct antiviral effects mediated by the NK cells (Alter et al., 2011),
and HIV-infected T cells can be directly susceptible to lysis by NK
cells (Ruscetti et al., 1986). On the other hand, some KIR that
correlate with better prognosis of HIV are those with negative
signaling capacity (Alter et al., 2009; Martin et al., 2002; Jennes
et al., 2006), and negatively signaling KIR have also been associated
with resolution of hepatitis C virus infection (Khakoo et al., 2004).
These findings about the negatively signaling KIR may be consistent
with the concept that the suppression of NK cell activity by negat ive
signaling NKR would allow for greater T cell responses to control
the infection. In fact, an inverse relationship was found between NK
cell responses and HIV-specific T cell responses in a cohort of elite
HIV controllers (Tomescu et al., 2012).
It has also been speculated that higher innate host responses,
such as those involving IFN, a potent activator of NK cells, might
contribute to differences in viral load set points that characterize
HIV patients (Carrington and Alter, 2012), and that this set point
R.M. Welsh, S.N. Waggoner / Virology 435 (2013) 37–4542
Page 6
decision may thus be made very early in infection. HIV RNA triggers
more IFN through TLR7/8 in DC from women than from men, and
women tend to have lower HIV set points (Meier et al., 2009).
Likewise, the development of AIDS-like disease in SIV-infected
macaques is associated with a prolonged type 1 IFN inflammatory
response that is substantially curtailed during the non-pathogenic
SIV infections of sooty mangabey and African green monkeys
(Bosinger et al., 2009; Jacquelin et al., 2009). These findings are
consistent with studies in the LCMV model showing that the
functions of NK cells activated during the first three days of
infection could have long term consequences on the levels and
duration of viral persistence (Waggoner et al., 2010, 2012).
Influence of NK cells on the generation of T cell memory
Because the magnitude of the T cell memory response is
proportional to the burst size of the acute response (Hou et al.,
1994), which is impaired by the activity of NK cells, it stands
to reason that NK cells should suppress vaccine efficiency and
the development of immunological memory. Indeed, depletion of
NK cells by administration of diphtheria toxin (DT) to mice
expressing the DT receptor on NK cells or in wild type mice
depleted of NK cells with anti-NK1.1 prior to immunization with
ovalbumin (OVA) resulted in the generation of considerably
higher numbers of OVA-specific transgenic T cells, which pro-
tected against an OVA-expressing tumor line (Soderquest et al.,
2011; Walzer et al., 2007). Correspondingly, the frequencies of
OVA-specific memory cells after immunization with an OVA-
expressing strain of Listeria monocytogenes were reduced in
NKp46 mutant mice harboring hyperactive NK cells (Narni-
Mancinelli et al., 2012). Our own studies have found increased
CD8 memory cell frequencies to LCMV and to Pichinde virus in NK
cell-depleted mice (unpublished). Collectively these results illus-
trate a dynamic equilibrium between NK cells and T cells, and it is
possible that suppression of the NK cell response might lead to
more effective vaccination. Many adjuvants are strong stimula-
tors of cytokines such as IFN, which can both activate NK cells and
promote T cell proliferation. Under some conditions, however, T
cell proliferation can be greatly reduced by IFN (Marshall et al.,
2011), indicating that the timing of these stimulating events may
be important to consider for optimal immunization.
Conclusions
Theimmunesystemistheonlyorgansysteminthebodythat
can expand and contract several-fold all within a few days, and it is
therefore very important to have mechanisms in place to keep
things from getting out of control and causing toxic shock-
like conditions, autoimmunity, or leukemias and lymphomas. As a
result, macrophages and regulatory T cells may make suppressive
cytokines like TGF
b
and IL-10 to restrain responses extrinsically,
and t he T cells themselves can intrinsically restrain their responses
by undergoing apoptosis or clonal exhaustion through the up-
regulation of inhibitory molecules (Wherry, 2011). It is now clear
that NK cells are another cell type that can regulate T cell responses,
and this regulation of T cells may be particularly strong under the
conditions of viral infections, which induce NK cell-activating
cytokines like IFN, IL-12, and IL-15. We showed here that the NK
cell response early in viral infections can have lasting effects on
viral pathogenesis and persistence. We speculate that prophylactic
depletion of NK cells may enhance vaccination and that therapeutic
depletion of NK cells in the midst of a persistent infection may
resurrect T cell responses and reduce viral titers.
Acknowledgments
This work was supported by NIH Training Grant T32 AI07349
and an Ellison Medical Foundation grant to SNW and by NIH
research grants AI017672, CA34461, AI081675, and AI046629 to
RMW. The views expressed are those of the authors and are not
necessarily the views of the NIH. We thank Keith Daniels for help
in the preparation of the figures.
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    • "Altogether , this suggests that the naturally tolerogenic environment of epithelia controls inappropriate NK cell functions and reactivity in steady conditions. Many studies have shown that NK cells impair T cell responses after systemic immunization or viral infection [39, 40]. Several mechanisms whereby NK cells regulate T cell responses were reported including engagement of the activating receptor NKG2D, TRAIL-dependent apoptosis , or cytokine production by NK cells [25, 37,[41][42][43][44][45][46][47]. "
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    • "The priming of Th1 cells is crucial for the generation and expansion of cytotoxic T lymphocytes (CTLs) and survival of CD8 + memory T cells, which are in turn able to kill infected or cancer cells (Kennedy and Celis 2008). NK cells also serve an important role in regulating DCmediated adaptive immune responses (Kalinski and others 2005a; Marcenaro and others 2006; Welsh and Waggoner 2013). Classically, NK cells are not only involved in the direct early control of viral infections and tumor immunosurveillance (Smyth and others 2002; Andrews and others 2003; Lee and others 2007) but NK cells also indirectly provide help by interacting with DCs at the site of inflammation or in the lymph nodes (Moretta 2002). "
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    • "Natural killer (NK) cells are a subset of innate lymphoid cells that function as early controllers of viral infections (Welsh and Waggoner, 2013). Recently the roles of NK cells in restricting the induction of adaptive anti-viral T cell responses and abetting the development of exhaustion have been highlighted (Fig. 2 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Although robust and highly effective anti-viral T cells contribute to the clearance of many acute infections, viral persistence is associated with the development of functionally inferior, exhausted, T cell responses. Exhaustion develops in a step-wise and progressive manner, ranges in severity, and can culminate in the deletion of the anti-viral T cells. This disarming of the response is consequential as it compromises viral control and potentially serves to dampen immune-mediated damage. Exhausted T cells are unable to elaborate typical anti-viral effector functions. They are characterized by the sustained upregulation of inhibitory receptors and display a gene expression profile that distinguishes them from prototypic effector and memory T cell populations. In this review we discuss the properties of exhausted T cells; the virological and immunological conditions that favor their development; the cellular and molecular signals that sustain the exhausted state; and strategies for preventing and reversing exhaustion to favor viral control. Copyright © 2014 Elsevier Inc. All rights reserved.
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