MHC Class I Cross-Presentation by Dendritic Cells Counteracts Viral Immune Evasion

Abstract

DCs very potently activate CD8⁺ T cells specific for viral peptides bound to MHC class I molecules. However, many viruses have evolved immune evasion mechanisms, which inactivate infected DCs and might reduce priming of T cells. Then MHC class I cross-presentation of exogenous viral Ag by non-infected DCs may become crucial to assure CD8⁺ T cell responses. Although many vital functions of infected DCs are inhibited in vitro by many different viruses, the contributions of cross-presentation to T cell immunity when confronted with viral immune inactivation in vivo has not been demonstrated up to now, and remains controversial. Here we show that priming of Herpes Simplex Virus (HSV)-, but not murine cytomegalovirus (mCMV)-specific CD8⁺ T cells was severely reduced in mice with a DC-specific cross-presentation deficiency. In contrast, while CD8⁺ T cell responses to mutant HSV, which lacks crucial inhibitory genes, also depended on CD8α⁺ DCs, they were independent of cross-presentation. Therefore HSV-specific CTL-responses entirely depend on the CD8α⁺ DC subset, which present via direct or cross-presentation mechanisms depending on the immune evasion equipment of virus. Our data establish the contribution of cross-presentation to counteract viral immune evasion mechanisms in some, but not all viruses.

Full text

ORIGINAL RESEARCH ARTICLE
published: 26 November 2012
doi: 10.3389/fimmu.2012.00348
MHC class I cross-presentation by dendritic cells
counteracts viral immune evasion
Katrin Nopora1‡, Caroline A. Bernhard1‡, Christine Ried1, Alejandro A. Castello1†, Kenneth M. Murphy2,
Peggy Marconi 3, Ulrich Koszinowski 4and Thomas Brocker1*
1Institute for Immunology, Ludwig-Maximilians-University Munich, Munich, Germany
2Department of Pathology and Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO, USA
3Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy
4Max von Pettenkofer-Institut, Ludwig-Maximilians-University Munich, Munich, Germany
Edited by:
Peter M.Van Endert, Université Paris
Descartes/INSERM, France
Reviewed by:
Sven Burgdorf, Rheinische
Friedrich-Wilhelms-Universität,
Germany
Marianne Boes, University Medical
Centre Utrecht, Netherlands
*Correspondence:
Thomas Brocker, Institute for
Immunology, Ludwig-Maximilians-
University Munich, Goethestraße 31,
80336 München, Germany.
e-mail: tbrocker@med.
uni-muenchen.de
†Present address:
Alejandro A. Castello, Universidad
Nacional de Quilmes, Buenos Aires,
Argentina.
‡Katrin Nopora and
Caroline A. Bernhard contributed
equally to this paper and share the
first authorship.
DCs very potently activate CD8+T cells specific for viral peptides bound to MHC class
I molecules. However, many viruses have evolved immune evasion mechanisms, which
inactivate infected DCs and might reduce priming of T cells. Then MHC class I cross-
presentation of exogenous viral Ag by non-infected DCs may become crucial to assure
CD8+T cell responses. Although many vital functions of infected DCs are inhibited in vitro
by many different viruses, the contributions of cross-presentation toT cell immunity when
confronted with viral immune inactivation in vivo has not been demonstrated up to now,
and remains controversial. Here we show that priming of Herpes Simplex Virus (HSV)-
, but not murine cytomegalovirus (mCMV)-specific CD8+T cells was severely reduced
in mice with a DC-specific cross-presentation deficiency. In contrast, while CD8+T cell
responses to mutant HSV, which lacks crucial inhibitory genes, also depended on CD8α+
DCs, they were independent of cross-presentation.Therefore HSV-specific CTL-responses
entirely depend on the CD8α+DC subset, which present via direct or cross-presentation
mechanisms depending on the immune evasion equipment of virus. Our data establish
the contribution of cross-presentation to counteract viral immune evasion mechanisms in
some, but not all viruses.
Keywords: dendritic cells, cross-priming, immune evasion
INTRODUCTION
Many viruses utilize a diversity of mechanisms to evade the
immune system (Tortorella et al., 2000;Yewdell and Hill, 2002).
Especially herpesviruses are extremely potent immune evaders and
Herpes Simplex Virus (HSV) shuts down host cell transcription,
RNA splicing, and protein synthesis by expressing an arsenal of
viral proteins (Hardy and Sandri-Goldin, 1994;Hill et al., 1995;
Spencer et al., 1997;Song et al., 2001;Smiley, 2004). HSV-1 infects
DCs with high efficiency as part of its life cycle (Coffin et al.,
1998;Salio et al., 1999;Kruse et al., 2000;Mikloska et al., 2001).
Consequently, infected DCs are severely compromised and fail to
mature, do not upregulate expression of MHC and other surface
molecules, cannot produce cytokines nor migrate properly (Salio
et al., 1999;Kruse et al., 2000;Samady et al., 2003;Prechtel et al.,
2005). As a consequence, HSV-infected DCs are non-functional
and cannot efficiently prime naive T cells in vitro (Salio et al.,
1999;Kruse et al., 2000).
It has been speculated that non-infected fully functional
bystander DCs could cross-present exogenous viral Ag derived
from infected and dying cells to secure priming of virus-
specific CD8+T cells (Heath and Carbone, 2001;Jirmo et al.,
2009). By experimentally excluding infection of DCs, it was
demonstrated that cross-presentation is principally sufficient to
mediate anti-viral CD8+T cell responses (Sigal et al., 1999;Nor-
bury et al., 2001). The selective capacity of CD8α+DCs to take
up dead cells (Iyoda et al., 2002) and to cross-prime CD8 T cells
(den Haan et al., 2000) suggests, that this DC subset may be espe-
cially important in anti-viral immunity. Indeed, several studies
have found CD8α+DCs to present viral Ag to CD8 T cells, when
mice were infected with HSV-1, lymphocytic choriomeningitis
virus, vaccinia virus, influenza virus, or respiratory syncytial virus
(Allan et al., 2003;Smith et al., 2003;Belz et al., 2004a,b, 2005;
Bedoui et al., 2009;Jirmo et al., 2009;Lukens et al., 2009) and
ablation of CD8α+DCs in Batf-3-deficient mice abrogated T cell
responses to west nile virus completely (Hildner et al., 2008).
However, experiments with murine cytomegalovirus (mCMV)
and viral mutants lacking immune evasion genes have not revealed
substantial differences in CD8 T cell responses, leaving the ques-
tion on the role of cross-presentation in the situation of viral
immune evasion open (Gold et al., 2004;Munks et al., 2007). In
the present study we compare immune evasion-competent HSV
and mCMV with their respective mutant forms lacking inhibitory
genes. We demonstrate that CD8 T cell responses to all wt and
mutant viruses studied depend on CD8α+DCs, which perform
www.frontiersin.org November 2012 | Volume 3 | Article 348 | 1

Nopora et al. Cross-priming enhances generation of virus-specific CTL
both, direct and cross-presentation, depending on the grade of
inhibition of virus infected DCs.
RESULTS
To study the extent to which CD8α+cross-presenting DCs can
compensate immune evasion, we utilized the KOS wild type
(wt) strain HSVwt, and its mutant (mut) derivative HSVmut
(∆UL41,ICP4,22,27), which lacks four viral genes responsible
for different viral “immune evasion”-strategies (Krisky et al.,
1998;Lauterbach et al., 2004). Vhs, the virion host shutoff protein
encoded by the gene UL41 causes destabilization and degrada-
tion of infected host cell mRNAs and is among other effects
responsible for down regulation of MHC I synthesis and expres-
sion (Hill et al., 1994;Tigges et al., 1996;Hinkley et al., 2000;
Koppers-Lalic et al., 2001). ICP4 reduces the stability of host cell
mRNA (Mogensen et al., 2004), ICP22 modifies the host RNA
polymerase II (Rice et al., 1995), while ICP27 inhibits mRNA bio-
genesis leading to shutoff of host protein synthesis (Hardwicke and
Sandri-Goldin, 1994;Hardy and Sandri-Goldin, 1994). Deletion
of these viral genes rescued maturation and immune functions of
infected human monocyte-derived DCs in vitro (Samady et al.,
2003). To confirm the effect of these viral genes on capacities of
murine DCs to prime CD8+T cells in vitro, we infected bone
marrow derived DCs (BMDCs) with wt HSVwt or HSVmut and
utilized them as APC for naïve CD8 T cells. The priming of naïve
HSV-glycoproteinB (gB)-specific CD8+T cells by HSVwt-infected
BMDCs was extremely inefficient and only at the highest DC: T
cell-ratio very few divided cells could be detected (Figure 1A). In
contrast, when BMDCs were infected with HSVmut, they were
highly efficient to prime T cells to divide (Figure 1A). To exclude
the possibility that these results were caused by different amounts
of viral gB-antigen being expressed by cells infected with the
different variants of HSV, we loaded infected DCs with titrated
amounts of an HSV-irrelevant LCMVgp33-41 peptide and tested
their capacity to prime specific CD8+P14 T cells (Figure 1B).
HSVmut-infected DCs were as efficient in priming naïve P14
T cells as non-infected mature DCs (Figure 1B). In contrast,
HSVwt-infected DCs needed 104times higher peptide concen-
trations to induce comparable CD8+T cell priming (Figure 1B).
These results show that deletion of these viral genes can rescue the
capacity of infected DCs to prime CD8+T cells in vitro.
Next we tested, if these different effects of wt- and mut-viruses
on DCs were of relevance for in vivo-priming of virus-specific
CTL and investigated the role of CD8α+DCs. Comparative stud-
ies between wt virus and mutant variants are generally difficult
to interpret due to different and partially uncharacterized viral
properties. Therefore we compared in the following studies only
CD8+T cell responses to same virus, either wt or mut, but in dif-
ferent mouse strains. To study the role of CD8α+DCs, we infected
Batf3−/−mice, which lack the transcription factor Batf3 leading to
DC (mock)
DC (HSVwt)
DC (HSVmut)
0
10
20
30
40
50
60
70
80
90
% divided cells
1/80 1/40 1/20
DC : T cell ratio
CFSE
+ DC (HSVwt) + DC (HSVmut)
gBT-1A
+ DC
CFSE
+ DC (HSVwt)
+ LCMVgp33-41
+ DC (HSVmut)
+ LCMVgp33-41
P14
+ DC
+ LCMVgp33-41
DC (mock)
DC (HSVwt)
DC (HSVmut)
% dividing cells
peptide (ng/ml)
0
10
20
30
40
50
60
70
80
90
100
010
-3 10
-1 10
110
3
B
cell numbercell number
FIGURE 1 | Deletion of HSV-1 immune evasion genes rescues
APC-functions of infected DCs in vitro.BMDC were infected with
HSVwt, HSVmut, or mock-infected and utilized to stimulate CFSE-labeled
TCR-transgenic HSVgB-specific CD8+gBT-I T cells (A). In addition DCs
were loaded with the LCMVgp33-41 peptide at the indicated
concentration and cultured with CFSE-labeled TCR-transgenic
gp33-41-specific CD8+P14 T cells (B). Appropriate conditions for DC:
Tcell ratio and peptide concentration were determined by titration [(A,B)
side insets]. Main graphs show results of CFSE-profiles of gated CD8+T
cells for one experiment out of four with similar results in which 2500
DCs were cultured with 5×104CFSE-labeledT cells for 4 days and
peptide concentration was 0.1 ng/ml.
Frontiers in Immunology | Antigen Presenting Cell Biology November 2012 | Volume 3 | Article 348 | 2

Nopora et al. Cross-priming enhances generation of virus-specific CTL
defective development of CD8α+and CD103+CD11b−DCs, the
two major cross-presenting DC-subpopulations (Hildner et al.,
2008;Edelson et al., 2010). As HSVmut does not interfere effi-
ciently with immune functions of infected DCs (Figures 1A,B),
we speculated that CD8+T cell responses against this viral
mutant should be more independent of cross-presentation than
those induced by HSVwt. While C57BL7/6 mice mounted strong
HSVgB-specific CD8+T cell responses as measured with the
respective MHC-tetramers, we could not detect significant gB-
specific CD8+T cells in HSVwt-infected Batf3−/−-mice at all
(Figure 2, upper panel). Surprisingly, also HSVmut, despite of
being devoid of major inhibitory genes, could not elicit gB-specific
T cell responses in Batf3−/−-mice, while it did so very efficiently
in wt mice (Figure 2, upper panel). When the spleens of the same
animals were monitored for IFN-γ-producing CD8+T cells, only
very low frequencies of IFN- γ+CD8+T cells were detectable
in HSVwt-infected Batf3−/−-mice (Figure 2, lower panel). IFN-
γ-producing CD8+gB-specific CD8+T cells were not present in
HSVmut-infected mice at all (Figure 2, lower panel). This data
indicates that the DC-subpopulations lacking in Batf3−/−-mice
are central for inducing CD8+T cell responses upon infection
with HSVwt and HSVmut.
In order to discriminate between the contributions of direct vs.
cross-presentation we next infected CD11c-Rac mice with HSV
viruses. In DCs of these mice, the dominant negative N17Rac-
transgene causes inhibition of uptake of exogenous soluble, cel-
lular, or apoptotic antigen leading to strong reduction of cross-
presentation (Kerksiek et al., 2005). As a consequence, CD11c-Rac
mice show deficient peripheral tolerance to self proteins (Luck-
ashenak et al., 2008), absent CD8 T cell responses to extracellu-
lar bacteria, to cell associated proteins, to apoptotic, or soluble
protein antigens (Kerksiek et al., 2005;Neuenhahn et al., 2006;
Luckashenak et al., 2008). Upon infection with HSVwt-virus we
could detect approximately half the frequency and numbers of
HSVgB-specific CD8+T cells in spleens of cross-presentation-
defective CD11c-Rac mice, as compared to non-transgenic lit-
termates (Figure 3A). Both, detection of specific T cells by
MHCI-tetramers (Figure 3A, upper panel), as well as analysis
of IFN-γ-producing CD8+T cells upon restimulation with viral
HSVgB-peptide in vitro (Figure 3A, lower panel) revealed strongly
reduced frequencies and total numbers of HSV-specific CD8+
T cells in CD11c-Rac mice. These differences were highly sig-
nificant, also when data from several independent experiments
were pooled (Figure 3B). In marked contrast, infection with
HSVmut elicited comparable frequencies and total amounts of
HSVgB-specific IFN-γ-producing CD8+T cells in CD11c-Rac-
and control mice (Figure 3C). These data indicate, that lack of
inhibitory genes in HSVmut allows normal priming of HSVgB-
specific CD8+T cells independently of cross-priming in CD11c-
Rac mice. It has been speculated that CD8+DCs preferentially
capture dying cells (Iyoda et al., 2002) and efficiently cross-present
relatively high-dose tissue-associated antigens (Kurts et al., 1998)
as probably occur during viral infections. To test if dependency
on cross-presentation was observed also upon inoculation with
low doses of virus, we infected mice with graded amounts of
HSVwt or HSVmut (Figure 3D). Our data indicate that indepen-
dently of the amount of virus utilized for infection, the priming
of approximately half of all HSV-specific CD8+T cells depends
on cross-presentation, as the responses to infection with HSVwt
were reduced approximately 50% in CD11c-Rac mice (Figure 3D).
However, when viral immune evasion genes were deleted in HSV-
mut, the amount of primed HSVgB-specific CD8+T cells in
CD11c-Rac mice was indistinguishable from those observed in
wt mice (Figure 3D).
We next tested if murine CMV (mCMVwt) and its triple
mutant mCMVmut, which is devoid of several MHC class I
0 1021031041050 10210 3104105
0 1021031041050 10210 3104105
0 102103104105
0
102
103
104
105
control control Batf3-ko control Batf3-ko
HSVwt HSVmut
gB Tetramer
CD44
0
1
2
3
4
5
6
gBTet+ CD8+ T cells (%)
control
Batf3-ko
HSVwt HSVmut
0
2.5
5.0
7.5
10
gBTet+ CD8+ T cells (x 10-5)
HSVwt HSVmut
0 1021031041050 10210 3104105
0 102103104105
0 102103104105
0 102103104105
0
102
103
104
105
IFN-γ
CD8
IFN-γ+ CD8+ T cells (%)
HSVwt HSVmut
0
1
2
3
4
5
6
7
**
-
--
no virus
FIGURE 2 | Batf3-ko mice cannot mount HSV-specific CD8 T cell
responses. Batf3-ko and C57BL/6-mice were infected i.v. with 4×106
infectious particles of HSV. Mice were sacrificed 5days later and spleens
were analyzed for presence of HSVgB-specific CD8+T cells with
H-2 Kb/gB498-505-tetramers (top panel) or by ex vivo restimulation with gB498-505
peptide and subsequent intracellular FACS-analysis for IFNγ-production (lower
panel). Dot plots are gated on CD8+T cells (upper panel) or lymphocytes
(lower panel) first (not shown). Data shown as bar graphs are mean±SEM
from n=3 mice per group. (**P=0.0018 as compared to unstimulated
control). Open bars represent C57BL/6-wild type mice and filled bars
represent Batf3-ko mice.This experiment has been repeated twice with
similar outcome.
www.frontiersin.org November 2012 | Volume 3 | Article 348 | 3

Nopora et al. Cross-priming enhances generation of virus-specific CTL
0
1
2
3
4
5
0
1
2
3
gBTet+ CD8+ T cells (%)
gBTet+ CD8+ T cells (x 10-5)
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0
0.5
1.0
1.5
2.0
2.5
HSVwt
gBTet+ CD8+ T cells (x 10-5)
gBTet+ CD8+ T cells (%)
*** *
0
50
100
150
% gBTet+ of CD8+ T cells
(relative to B6)
control
gB Tetramer
control CD11c-Rac
CD44
IFN-γ
CD8
010 21031041050 10 21031041050 10210 3104105
0
102
103
104
105
0.0
2.5
5.0
7.5
10.0
control
CD11c-Rac
IFN-γ+ CD8+ T cells (%)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
IFN-γ+ CD8+ T cells (%)
*
0 10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
0 10 210 310 410 5
0
10 2
10 3
10 4
10 5
0 10 210 310 410 5
0 10 210 310 410 5
gB Tetramer
CD44
IFN-γ
CD8
HSVwt
control control CD11c-Rac
HSVmut
control
CD11c-Rac
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
control
CD11c-Rac
IFN-γ+ CD8+ T cells (%)
0.4 4 40
HSVwt titers (1x105)
0
*
*
*
0.0
2.5
5.0
7.5
10.0
control
CD11c-Rac
IFN-γ+ CD8+ T cells (%)
0.4 4 40
HSVmut titers (1x105)
0
AB
C
HSVwt
HSVwt
HSVmut
HSVmut
HSVmut-
D
-
-
-
--
control
CD11c-Rac
no virus
no virus
***
FIGURE 3 | Cross-presentation deficient CD11c-Rac mice mount
reduced CD8T cell responses to HSVwt, but normal responses to
immune evasion-deficient HSVmut. Mice were immunized i.v. with
4×106infectious particles (A,B,C) or graded amounts (D) of HSVwt or
HSVmut and 5 days later CD8+T cells from spleens were analyzed as
described in Figure 2. Dot plots are gated on CD8+T cells [(A,C) upper
panel] or all lymphocytes [(A,C) lower panel] (not shown). Data from one
out of four experiments with similar results (n=3 mice per group) are
shown as mean ±SEM in bar graphs (a, c; side insets), t-test analysis, a,
upper panel: ***P<0.0001; *P=0.0284; a, lower panel *P=0.0243. (B)
Pooled data from seven experiments (n=3 mice per group, total n=21)
are displayed as percent of control (C57BL/6).The mean of each control
group was set to 100% and the data from the CD11c-Rac-groups were
calculated relative to the respective control group (***P=0.0001). (D)
Mice were immunized with the indicated amounts of HSVwt or HSVmut.
Analyses were performed as described in (A). HSVwt 0.4×105,
*P=0.0221; 4 ×105, *P=0.0223; 40 ×105, *P=0.0243; One out of two
experiments with similar outcome is shown (n=3 mice per group).
Frontiers in Immunology | Antigen Presenting Cell Biology November 2012 | Volume 3 | Article 348 | 4

Nopora et al. Cross-priming enhances generation of virus-specific CTL
inhibitory genes would be similarly dependent on cross-presenting
CD8α+DCs. The mutant form of mCMVwt (C3X), mCMVmut
(∆m04∆m06∆m152; Wagner et al., 2002), lacks three known
“immunoevasins” that act on the MHC class I presentation path-
way. While m04 binds to MHC class I (Kleijnen et al., 1997)
and prevents activation of T cells (Kavanagh et al., 2001), m06
retargets MHC class I to lysosomal degradation (Reusch et al.,
1999) and m152 retains MHC I/peptide complexes in the ER-
cis-Golgi compartment (Ziegler et al., 1997) leading to an over-
all reduction of MHC class I surface expression. Accordingly,
when mCMVwt- or mCMVmut-infected gp33-41 peptide-pulsed
BMDC were cocultured with gp33-41 peptide-specific P14 T
cells, the mCMVmut-infected DCs induced more efficient T cell
responses as compared to DCs infected with mCMVwt. How-
ever, in comparison to the HSV study (Figures 1A,B), the over-
all inhibitory effect of mCMVwt virus was weaker as compared
to HSV (Figure 4A).
In analogy to the experiments with HSV described above, we
next infected wt and Batf3-ko mice with the two viruses and mea-
sured IFN-γproducing virus-specific CD8 T cells. CTL-responses
against both, mCMVwt and mCMVmut, entirely depended on
the presence of CD8α+DCs, as we could not detect mCMV-
specific CD8 T cells in Batf-3 ko mice immunized with either
virus (Figures 4B,C). To further analyze if mCMV-specific CD8 T
cells would be primed via direct or cross-presentation by CD8α+
DCs, we infected CD11c-Rac mice and compared the responses to
those elicited in non-transgenic littermates (Figure 4D). How-
ever, and in contrast to the results obtained with HSVwt and
HSVmut, mCMV-specific cytotoxic T cells were indistinguish-
able between wt and CD11c-Rac mice for mCMVwt as well as
mCMVmut (Figure 4E). Therefore we conclude that priming of
mCMV-specific T cells crucially depends on CD8α+DCs, but is
independent of their cross-priming capacities.
DISCUSSION
CD8α+DCs are important for immunity as they have the spe-
cific ability to produce high levels of IL-12, direct Th1 responses
and produce CD8+T cell responses due to their capacity to cross-
present exogenous Ag (Shortman and Heath, 2010). In our study
we investigated the interdependency of CD8α+DCs, cross- and
direct presentation with viral inhibition of immune functions.
Such functionally inhibitory immune subversion mechanisms,
which affect DCs, have been identified for HSV and mCMV
and were deleted in the respective mutant strains selected for
the present study. A key factor for HSV with respect to inhibi-
tion of DC-functions is vhs, which inhibits expression of MHC
class I, class II, cytokine and chemokine production (reviewed
in Smiley, 2004). Accordingly, vhs-null mutations show severely
impaired viral pathogenesis and replication in mouse models
in vivo (Strelow and Leib, 1996;Leib et al., 1999), suggesting
that vhs is a virulence factor of HSV causing defects of DC-
functions and host immunity (reviewed in Smiley, 2004). We find
that DCs infected with HSVmut can very well prime CD8+T
cells in vitro. This finding suggests that also in vivo HSVmut-
infected cells should be able to prime CD8 T cells directly and
do not depend on cross-presentation by non-infected bystander
DCs. Our interpretation is corroborated by the finding that in
CD11c-Rac mice, which have reduced cross-priming capacities,
CD8 T cell responses to HSVmut are normal, and we conclude
that they do not depend on cross-priming. As HSVmut-infected
Batf3−/−-mice do not mount HSV-specific CD8 T cell-responses
in vivo, we assume that the DC-subpopulations absent in these
mice are responsible for direct HSV-presentation. In contrast,
HSVwt-infected DCs are severely inhibited to prime CD8 T cells
in vitro, HSVwt-induced CD8 T cell responses are also absent in
Batf3−/−-mice and do depend on cross-presenting DCs, as they
are strongly reduced in CD11c-Rac mice. A potential caveat to this
explanation could be the fact, that CD11c-Rac mice do not only
have a defect in endocytosis and cross-presentation, but also have
slightly lower numbers of CD8α+DCs in their spleens (Kerksiek
et al., 2005). It is therefore theoretically possible, that the reduced
T cell responses rather reflect the reduced numbers of CD8α+
DCs than their capacities to cross-present. However, this explana-
tion is unlikely, as responses to HSVmut are identical in both, wt
and CD11c-Rac mice, indicating sufficient DC numbers for the
induction of optimal CTL-responses.
In contrast, HSVwt-infected DCs can still prime CD8+T
cells in vitro (Figure 1), albeit with much lowered efficacy.
Such “residual” DC-activity could account for the lower T cell
response measured in HSVwt-infected CD11c-Rac mice, when
cross-presentation is suboptimal. Our results therefore suggest,
that (i) CD8α+DCs are responsible for both, direct and cross-
priming of HSV-specific CD8+T cells and (ii) that cross-
priming becomes more important when the functional inhibition
of infected DCs increases. Cross-priming can compensate viral
immune evasion very efficiently. As a consequence, CD8 T cell
responses to HSVwt and HSVmut become indistinguishable in
cross-presentation competent mice.
This observation seems to parallel earlier findings with other
herpesviruses such as mCMV, where deletion of genes showed lit-
tle or no impact on T cell responses in wt mice in vivo (Gold
et al., 2004;Munks et al., 2007). In the past it has therefore been
difficult to establish a role for cross-presentation by using viruses
and their specific mutants. For example, removal of mCMV genes
affecting MHC class I expression had no effect on the CD8+T
cell responses in vivo (Munks et al., 2007). The interpretation
of these findings were that either directly infected DCs can still
prime CD8+T cells efficiently despite viral inhibition, or that
cross-priming is so potent that it dominates CD8+T cell priming
to mCMV (Snyder et al., 2010), making responses to mutant and
wt virus similar (Munks et al., 2007). It has also been discussed
that responses induced by mCMVwt and mutant strains in pre-
vious studies were similar, because in addition to inhibition of
MHC class I presentation, mCMV is known to also inhibit other
functions of infected DCs. For example shutting off expression of
costimulatory molecules and upregulating inhibitory ligands by
mCMV results in failure to prime CD8+T cells in vitro (Andrews
et al., 2001;Loewendorf et al., 2004;Benedict et al., 2008).However,
these inhibitory mechanisms of mCMV have not been entirely
identified yet and therefore the responsible viral genes were nei-
ther deleted in the respective experiments (Munks et al., 2007)
nor in our study. In a recent publication, Torti et al. (2011)
analyzed primary and memory CTL-responses to a single dele-
tion mCMV-mutant (mCMV∆m157) in Batf3−/−-mice. While,
www.frontiersin.org November 2012 | Volume 3 | Article 348 | 5

Nopora et al. Cross-priming enhances generation of virus-specific CTL
m139(T8L)
M45(H9I)
M57(S9V)
0
1
2
3
0
1
2
3
IFN-γ+ CD8+ T cells (%)
IFN-γ+ CD8+ T cells (%)
B6 Batf3-ko
mCMVwt
B6 Batf3-ko
mCMVmut
IFN-γ
CD8
control
mCMVwt mCMVmut
m139 M45 M57
B6
Batf3-ko
B6
Batf3-ko
mCMVmut
0
1
2
3
4
mCMVwt
m139 M45 M57 m141 M38 -
0
1
2
3
4
5
6
7
B6
Rac
control
IFN-γ+ CD8+ T cells (%)
IFN-γ+ CD8+ T cells (%)
IFN-γ
CD8
m139
B6 Rac
mCMVwt
B6 Rac
mCMVmut
control
B
C
D
E
m139 M45 M57
m139 M45 M57 m141 M38 -
+ DC (mCMVwt)
+ LCMVgp33-41
+ DC (mCMVmut)
+ LCMVgp33-41
P14
+ DC
+ LCMVgp33-41
% dividing cells
0
10
20
30
40
50
60
70
80
90
100
010
-3 10
-1 10
110
3
DC (mock)
DC (mCMVwt)
DC (mCMVmut)
A
CFSE
cell number
peptide (ng/ml)
FIGURE 4 | Continued
Frontiers in Immunology | Antigen Presenting Cell Biology November 2012 | Volume 3 | Article 348 | 6

Nopora et al. Cross-priming enhances generation of virus-specific CTL
FIGURE 4 | mCMV responses do not depend on cross-presentation.
BMDC were infected with mCMVwt, mCMVmut, or mock-infected. DCs were
utilized to either stimulate 5 ×104CFSE-labeledTCR-transgenic
gp33-41-specific CD8+P14 T cells as described in Figure 1B (A). CFSE-profiles
of gated CD8+T cells are shown for the peptide concentration 0.1 ng/ml.
Graphs show results of gated divided T cells of one experiment out of two
with similar results. (B) Batf3-ko and C57BL/6-mice were infected i.v. with
4×106infectious particles of mCMVwt or mCMVmut. Mice were sacrificed
7 days later and spleens were analyzed for presence of mCMV-specific CD8+
T cells by ex vivo restimulation with the indicated mCMV-peptides and
subsequent intracellular FACS-analysis for IFNγ-production. Dot plots are
gated on lymphocytes first (not shown). (C) Data from (B) shown as bar
graphs are mean ±SEM from n=3 mice per group. Open bars represent
C57BL/6-wild type (wt) mice and filled bars represent Batf3-ko mice. This
experiment has been repeated twice with similar outcome. (D) CD11c-Rac- or
C57BL/6-mice were infected i.v. with 4 ×106infectious particles of mCMVwt
or mCMVmut. Mice were sacrificed 5 days later and spleens were analyzed
for presence of mCMV-specific CD8+T cells by ex vivo restimulation with the
indicated mCMV-peptides and subsequent intracellular FACS-analysis for
IFNγ-production. Dot plots are gated on lymphocytes first (not shown). (E)
Data from (D) shown as bar graphs are mean ±SEM from n=3 mice per
group. Open bars represent C57BL/6-wt mice, black bars represent Batf3-ko
mice, gray bars are non-immunized controls.This experiment has been
repeated twice with similar outcome.
similar to our findings, primary CD8 T cell responses to several
epitopes were impaired, development of memory T cells rather
seemed to depend on direct priming, as they were normal in
Batf3−/−-mice. Although mCMVwt exerts inhibitory effects on
DCs in vitro in our experiments, these effects did apparently not
affect CD8 T cell immunity in vivo. Different experimental strate-
gies have suggested that cross-presentation plays a dominant role
in CD8 T cell priming during viral infection (Wilson et al., 2006;
Snyder et al., 2010),but intravital microscopy could document also
direct priming of naive CD8 T cells by DCs infected with vaccinia
virus (Hickman et al., 2008), which also strongly inhibits DC-
functions (Engelmayer et al., 1999). The fact that the responses in
control and CD11c-Rac mice were comparable for both,mCMVw t
as well as mCMVmut, suggests that cross-presentation does not
contribute substantially to mCMV T cell immunity. Eventually,
mCMV-mediated inhibition of DCs is not sufficient to make
cross-presentation necessary. In contrast, the lack of responses in
Batf3−/−-mice indicates absolute necessity for (direct) presenta-
tion by CD8α+DCs. Accordingly, our findings that Batf3−/−-mice,
which are deficient for CD8α+DCs, cannot mount CD8+T cell
responses to neither HSV, HSVmut, mCMV, mCMVmut, under-
line that this DC-subpopulation is responsible for both – direct
priming upon infection and cross-presentation of viral material
from infected surrounding cells. However, our findings can even-
tually not be generalized for all viruses and routes of infection. For
example, the“natural” route of infection for HSV is via the skin or
mucosa, where other DC-subpopulations will be involved in direct
and cross-presentation. In addition, the deleterious effects of viral
immune evasion genes may be different in different DC subtypes.
Also the interplay of different DC-subpopulation in skin, mucosa
and the respective draining lymph nodes may be different from
the scenario in the spleen.
The roles direct vs. cross-priming play during viral infection
are certainly dependent on the type of virus, its cellular tropism
and the route of infection. While our results establish that cross-
presentation counteracts effects of viral inhibitory genes on DCs,
full understanding of the different contributions of direct vs. cross-
presentation will help to improve classical vaccination as well as
DC-targeting approaches.
MATERIALS AND METHODS
ANIMALS
CD11c-Rac- (Kerksiek et al., 2005), Batf3−/−- (Hildner et al.,
2008), P14- (Pircher et al., 1989), and gBT-1-mice (Mueller et al.,
2002) have been described before and were all maintained on the
C57BL/6 background. Mice were infected with virus preparation
diluted in 50 µl PBS and sacrificed with CO2at the indicated time
points. Mice were bred and housed at the animal facilities of the
Institute for Immunology (LMU, Munich, Germany) and treated
in accordance with established guidelines of the Regional Ethics
Committee of Bavaria. Animal protocols were approved by local
authorities.
VIRUSES AND INFECTIONS
HSVwt (KOS), HSVmut (∆UL41,ICP4,22,27), mCMVwt (C3X),
mCMVmut (∆m04∆m06∆m152) were prepared and utilized as
described previously (Wagner et al., 2002;Lauterbach et al., 2004).
Mice were infected intravenously (i.v.) with the amount of virus
indicated in the respective Figure legends.
CELL ISOLATION AND PURIFICATION
Single cell suspensions from spleens or lymph nodes were obtained
by mechanical disruption using a pestle followed by enzymatic
digestion in serum-free RPMI medium containing Liberase CI
(0.42 mg/ml) and DNase I (0.2 mg/ml, both from Roche, Basel,
Switzerland) for 20 min at 37˚C. Cells were passed through a
70 µm nylon mesh strainer. Cells were counted on a cell counter
(Beckman Coulter, Munich, Germany).
GENERATION OF BONE MARROW DERIVED DENDRITIC CELLS
Femurs and tibiae were flushed with IMDM and erythrocytes were
lysed by incubation in ACK buffer for 2 min at room temperature.
1×107bone marrow cells were plated in 10ml IMDM contain-
ing 10% heat inactivated FCS, 2 mM glutamine, 100 U/ml peni-
cillin, 100 µg/ml streptomycin sulfate, 50 µM 2-mercaptoethanol,
and 20 ng/ml GM-CSF (IMDM complete) in Petri-dishes. On
day three suspension cells and loosely adherent cells were dis-
lodged by gentle pipetting and adherent cells were subsequently
released by incubation in cold PBS containing 1 mM EDTA.
7.5 ×106cells were reseeded in 10 ml fresh IMDM complete per
10 cm dish.
INFECTION OF DC
For in vitro studies BMDCs were infected with virus as described
previously (Samady et al., 2003) and utilized 48 h post trans-
fection for T cell stimulation assays. DC (5 ×105) from day 7
BMDC-cultures were pelleted at 1,400 rpm for 5 min at room tem-
perature. The DC were then infected at a multiplicity of infection
www.frontiersin.org November 2012 | Volume 3 | Article 348 | 7

Nopora et al. Cross-priming enhances generation of virus-specific CTL
(MOI) of 1, unless otherwise stated, by resuspension in 200 ml
of IMDM medium containing 5 ×105PFU of virus for 1 h at
37˚C and 5% CO2. One milliliter of IMDM supplemented with
granulocyte-macrophage colony-stimulating factor (0.1 mg/ml)
was then added, and the DC were incubated at 37˚C and 5% CO2.
CFSE LABELING
P14 and gB-T1 T cells were purified from lymphocyte cell sus-
pensions by negative selection (CD8 T cell columns; R&D Sys-
tems, Minneapolis, MN, USA). For CFSE labeling 1–50 ×106
erythrocyte-free cells from lymph nodes and spleens were washed
twice with PBS and labeled with 5 µM CFSE (Molecular Probes,
Eugene, OR, USA) for 10 min at 37˚C in PBS. After stopping the
reaction (PBS, 2% FBS) and washing in PBS, cells were utilized for
the in vitro assays.
T CELL STIMULATION
5×104CFSE-labeled P14 and gB-T1 T cells were cultured for
4 days with 2500 DCs, which were either virus infected or loaded
with 1 µg/ml of the respective peptide.
ANTIBODIES AND FLOW CYTOMETRY
Lymphocytes were analyzed using anti-CD8a-APC, anti-CD44-
FITC, CD19-PerCP, IFNg-PE from Caltag (Burlingame, CA,
USA). H-2 Kb/gB498–505-tetramer-PE complexes were purchased
from ProImmune Limited. Staining of surface molecules
was performed with 1 ×106to 6 ×106cells in cold stain-
ing buffer for 30 min at 4˚C (15 min at room tempera-
ture in the dark when MHC multimers were used). Dead-
cell exclusion was attained by incubation with 1 µg/ml ethid-
ium monoazide bromide (EMA, Molecular Probes) prior to
surface staining or the addition of 0.8 mg/ml propidium iodide
(PI, Sigma). Intracellular staining for cytokines was performed
with the Cytofix/Cytoperm kit (PharMingen). Flow cytome-
try was performed with a FACSCalibur or FACSaria (Bec-
ton Dickinson), and data were analyzed with FlowJo software
(Tree Star).
STIMULATION OF CYTOKINE PRODUCTION BY EPITOPE-SPECIFIC T
CELLS
Splenocyte suspensions were prepared according to standard pro-
cedures and lymphocytes were stimulated with 1 µg/ml of the
indicated peptides in the presence of Brefeldin A (10 µg/ml;
Sigma, St. Louis, MO, USA). Cells were surface stained for 30min
at 4˚C before the fixation and permeabilization in 500 µl 2x
FacsLyse (BD Biosciences) containing 0.05% Tween 20 (Sigma-
Aldrich, St. Louis, MO, USA) for 10 min at room temper-
ature. The intracellular staining of cytokines was performed
for 30 min at room temperature. Analytic flow cytometry was
performed on a FACScanto II (Becton Dickinson). Analysis
was performed using FlowJo software (Tree Star, San Carlos,
CA, USA).
STATISTICAL ANALYSIS
All statistical analyses were performed using the two-tailed Stu-
dent’s t-test with unequal variance.
ACKNOWLEDGMENTS
We thank A. Bol and W. Mertl for assistance with animal
maintenance and experimentation. This work was supported by
the Deutsche Forschungsgemeinschaft (DFG) grant BR1889/5 to
Thomas Brocker.
REFERENCES
Allan, R. S., Smith, C. M., Belz, G. T.,
van Lint, A. L., Wakim, L. M.,Heath,
W., et al. (2003). Epidermal viral
immunity induced by CD8alpha+
dendritic cells but not by Langerhans
cells. Science 301, 1925–1928.
Andrews, D. M., Andoniou, C. E.,
Granucci, F., Ricciardi-Castagnoli,
P., and Degli-Esposti, M. A. (2001).
Infection of dendritic cells by
murine cytomegalovirus induces
functional paralysis. Nat. Immunol.
2, 1077–1084.
Bedoui, S., Whitney,P. G., Waithman, J.,
Eidsmo, L., Wakim, L., Caminschi,
I., et al. (2009). Cross-presentation
of viral and self antigens by skin-
derived CD103+dendritic cells.
Nat. Immunol. 10, 488–495.
Belz, G. T., Shortman, K., Bevan,
M. J., and Heath, W. R. (2005).
CD8alpha+dendritic cells selec-
tively present MHC class I-restricted
noncytolytic viral and intracellu-
lar bacterial antigens in vivo. J.
Immunol. 175, 196–200.
Belz, G. T., Smith, C. M., Eich-
ner, D., Shortman, K., Karupiah,
G., Carbone, F. R., et al. (2004a).
Cutting edge: conventional CD8
alpha+dendritic cells are generally
involved in priming CTL immu-
nity to viruses. J. Immunol. 172,
1996–2000.
Belz, G. T., Smith, C. M., Kleinert, L.,
Reading, P., Brooks, A., Shortman,
K., et al. (2004b). Distinct migrat-
ing and nonmigrating dendritic cell
populations are involved in MHC
class I-restricted antigen presenta-
tion after lung infection with virus.
Proc. Natl. Acad. Sci. U.S.A. 101,
8670–8675.
Benedict, C. A., Loewendorf, A., Gar-
cia, Z., Blazar, B. R., and Janssen, E.
M. (2008). Dendritic cell program-
ming by cytomegalovirus stunts
naive T cell responses via the PD-
L1/PD-1 pathway. J. Immunol. 180,
4836–4847.
Coffin, R. S., Thomas, S. K., Thomas, N.
S., Lilley,C. E.,Pizzey,A. R.,Griffiths,
C. H., et al. (1998). Pure populations
of transduced primary human cells
can be produced using GFP express-
ing herpes virus vectors and flow
cytometry. Gene Ther. 5, 718–722.
den Haan, J. M., Lehar, S. M., and
Bevan, M. J. (2000). CD8(+) but not
CD8(−) dendritic cells cross-prime
cytotoxic T cells in vivo. J. Exp. Med.
192, 1685–1696.
Edelson, B. T., Kc, W., Juang, R.,
Kohyama, M., Benoit, L. A.,
Klekotka, P. A., et al. (2010). Periph-
eral CD103+dendritic cells form
a unified subset developmentally
related to CD8alpha+conventional
dendritic cells. J. Exp. Med. 207,
823–836.
Engelmayer, J., Larsson, M., Subklewe,
M., Chahroudi, A., Cox, W. I., Stein-
man, R. M., et al. (1999). Vac-
cinia virus inhibits the maturation
of human dendritic cells: a novel
mechanism of immune evasion. J.
Immunol. 163, 6762–6768.
Gold, M. C., Munks, M. W., Wag-
ner, M., McMahon, C. W., Kelly,
A., Kavanagh, D. G., et al. (2004).
Murine cytomegalovirus interfer-
ence with antigen presentation has
little effect on the size or the effec-
tor memory phenotype of the CD8
T cell response. J. Immunol. 172,
6944–6953.
Hardwicke, M. A., and Sandri-Goldin,
R. M. (1994). The herpes simplex
virus regulatory protein ICP27 con-
tributes to the decrease in cellu-
lar mRNA levels during infection. J.
Virol. 68, 4797–4810.
Hardy, W. R., and Sandri-Goldin, R.
M. (1994). Herpes simplex virus
inhibits host cell splicing, and regu-
latory protein ICP27 is required for
this effect. J. Virol. 68, 7790–7799.
Heath, W. R., and Carbone, F. R. (2001).
Cross-presentation in viral immu-
nity and self-tolerance. Nat. Rev.
Immunol. 1, 126–134.
Hickman, H. D., Takeda, K., Skon, C.
N., Murray, F. R., Hensley, S. E.,
Loomis, J.,et al. (2008). Direct prim-
ing of antiviral CD8+T cells in
the peripheral interfollicular region
of lymph nodes. Nat. Immunol. 9,
155–165.
Hildner, K., Edelson, B. T., Purtha, W.
E., Diamond, M., Matsushita, H.,
Kohyama, M., et al. (2008). Batf3
deficiency reveals a critical role for
CD8alpha+dendritic cells in cyto-
toxic T cell immunity. Science 322,
1097–1100.
Frontiers in Immunology | Antigen Presenting Cell Biology November 2012 | Volume 3 | Article 348 | 8

Nopora et al. Cross-priming enhances generation of virus-specific CTL
Hill, A., Jugovic, P., York, I., Russ, G.,
Bennink, J., Yewdell, J., et al. (1995).
Herpes simplex virus turns off the
TAP toe vade host immunity. Nature
375, 411–415.
Hill, A. B., Barnett, B. C., McMichael,
A. J., and McGeoch, D. J. (1994).
HLA class I molecules are not trans-
ported to the cell surface in cells
infected with herpes simplex virus
types 1 and 2. J. Immunol. 152,
2736–2741.
Hinkley, S., Ambagala, A. P., Jones, C.
J., and Srikumaran, S. (2000). Avhs-
like activity of bovine herpesvirus-1.
Arch. Virol. 145, 2027–2046.
Iyoda, T., Shimoyama, S., Liu, K.,
Omatsu,Y.,Akiyama, Y.,Maeda, Y., et
al. (2002). The CD8+dendritic cell
subset selectively endocytoses dying
cells in culture and in vivo. J. Exp.
Med. 195, 1289–1302.
Jirmo, A. C., Nagel, C. H., Bohnen,
C., Sodeik, B., and Behrens, G. M.
(2009). Contribution of direct and
cross-presentation to CTL immu-
nity against herpes simplex virus 1.
J. Immunol. 182, 283–292.
Kavanagh, D. G., Gold, M. C., Wag-
ner, M., Koszinowski, U. H., and
Hill, A. B. (2001). The multiple
immune-evasion genes of murine
cytomegalovirus are not redundant:
m4 and m152 inhibit antigen pre-
sentation in a complementary and
cooperative fashion. J. Exp. Med.
194, 967–978.
Kerksiek, K. M., Niedergang, F.,
Chavrier, P., Busch, D. H., and
Brocker, T. (2005). Selective Rac1
inhibition in dendritic cells dimin-
ishes apoptotic cell uptake and
cross-presentation in vivo. Blood
105, 742–749.
Kleijnen, M. F., Huppa, J. B., Lucin,
P., Mukherjee, S., Farrell, H., Camp-
bell, A. E., et al. (1997). A mouse
cytomegalovirus glycoprotein, gp34,
forms a complex with folded class
I MHC molecules in the ER which
is not retained but is transported
to the cell surface. EMBO J. 16,
685–694.
Koppers-Lalic, D., Rijsewijk, F. A., Ver-
schuren, S. B., van Gaans-Van den
Brink, J. A., Neisig, A., Ressing, M.
E., et al. (2001). The UL41-encoded
virion host shutoff (vhs) protein
and vhs-independent mechanisms
are responsible for down-regulation
of MHC class I molecules by bovine
herpesvirus 1. J. Gen. Virol. 82,
2071–2081.
Krisky, D. M., Marconi, P. C., Olig-
ino, T. J., Rouse, R. J., Fink, D. J.,
Cohen, J. B., et al. (1998). Devel-
opment of herpes simplex virus
replication-defective multigene
vectors for combination gene
therapy applications. Gene Ther. 5,
1517–1530.
Kruse, M., Rosorius, O., Kratzer, F.,
Stelz, G., Kuhnt, C., Schuler, G.,
et al. (2000). Mature dendritic
cells infected with herpes simplex
virus type 1 exhibit inhibited T-
cell stimulatory capacity. J. Virol. 74,
7127–7136.
Kurts, C., Miller, J. F., Subramaniam,
R. M., Carbone, F. R., and Heath,
W.R. (1998). MHC class I-restric ted
cross-presentation is biased towards
high dose Ag and those released dur-
ing cellular destruction. J. Exp. Med.
188, 409–414.
Lauterbach, H., Kerksiek, K. M., Busch,
D. H., Berto, E., Bozac, A., Mavro-
mara, P., et al. (2004). Protection
from bacterial infection by a sin-
gle vaccination with replication-
deficient mutant herpes simplex
virus type 1. J. Virol. 78, 4020–4028.
Leib, D. A., Harrison, T. E., Laslo, K.
M., Machalek, M. A., Moorman, N.
J., and Virgin, H. W. (1999). Inter-
ferons regulate the phenotype of
wild-type and mutant herpes sim-
plex viruses in vivo. J. Exp. Med. 189,
663–672.
Loewendorf, A., Kruger, C., Borst,
E. M., Wagner, M., Just, U., and
Messerle, M. (2004). Identification
of a mouse cytomegalovirus gene
selectively targeting CD86 expres-
sion on antigen-presenting cells. J.
Virol. 78, 13062–13071.
Luckashenak,N., Schroeder, S., Endt, K.,
Schmidt,D.,Mahnke, K., Bachmann,
M. F., et al. (2008). Constitutive
cross presentation of tissue antigens
by dendritic cells controls CD8+T
cell tolerance in vivo. Immunity 28,
521–532.
Lukens, M. V., Kruijsen, D., Coenjaerts,
F. E., Kimpen, J. L., and van Bleek,
G. M. (2009). Respiratory syncytial
virus-induced activation and migra-
tion of respiratory dendritic cells
and subsequent antigen presenta-
tion in the lung-draining lymph
node. J. Virol. 83, 7235–7243.
Mikloska, Z., Bosnjak, L., and Cun-
ningham, A. L. (2001). Immature
monocyte-derived dendritic cells are
productively infected with herpes
simplex virus type 1. J. Virol. 75,
5958–5964.
Mogensen,T. H., Melchjorsen, J.,Malm-
gaard, L., Casola, A., and Paludan,
S. R. (2004). Suppression of proin-
flammatory cytokine expression by
herpes simplex virus type 1. J. Virol.
78, 5883–5890.
Mueller, S. N., Heath, W., McLain, J.
D., Carbone, F. R., and Jones, C. M.
(2002). Characterization of two TCR
transgenic mouse lines specific for
herpes simplex virus. Immunol. Cell
Biol. 80, 156–163.
Munks, M. W., Pinto, A. K., Doom,
C. M., and Hill, A. B. (2007). Viral
interference with antigen presenta-
tion does not alter acute or chronic
CD8 T cell immunodominance in
murine cytomegalovirus infection. J.
Immunol. 178, 7235–7241.
Neuenhahn, M., Kerksiek, K. M.,
Nauerth, M., Suhre, M. H., Schie-
mann, M., Gebhardt, F. E., et al.
(2006). CD8alpha+dendritic cells
are required for efficient entry of
Listeria monocytogenes into the
Spleen. Immunity 25, 619–630.
Norbury, C. C., Princiotta, M. F., Bacik,
I., Brutkiewicz, R. R., Wood, P.,
Elliott, T., et al. (2001). Multi-
ple antigen-specific processing path-
ways for activating naive CD8+
T cells in vivo. J. Immunol. 166,
4355–4362.
Pircher, H., Burki, K., Lang, R., Hen-
gartner, H., and Zinkernagel, R. M.
(1989). Tolerance induction in dou-
ble specific T-cell receptor trans-
genic mice varies with antigen.
Nature 342, 559–561.
Prechtel, A. T., Turza, N. M., Kobelt,
D. J., Eisemann, J. I., Coffin, R. S.,
McGrath, Y., et al. (2005). Infection
of mature dendritic cells with her-
pes simplex virus type 1 dramati-
cally reduces lymphoid chemokine-
mediated migration. J. Gen. Virol.86,
1645–1657.
Reusch, U., Muranyi, W., Lucin, P.,
Burgert, H. G., Hengel, H., and
Koszinowski, U. H. (1999). A
cytomegalovirus glycoprotein re-
routes MHC class I complexes to
lysosomes for degradation. EMBO J.
18, 1081–1091.
Rice, S. A., Long, M. C., Lam, V., Schaf-
fer, P. A., and Spencer, C. A. (1995).
Herpes simplex virus immediate-
early protein ICP22 is required for
viral modification of host RNA poly-
merase II and establishment of the
normal viral transcription program.
J. Virol. 69, 5550–5559.
Salio, M., Cella, M., Suter, M., and Lan-
zavecchia, A. (1999). Inhibition of
dendritic cell maturation by herpes
simplex virus. Eur. J. Immunol. 29,
3245–3253.
Samady, L., Costigliola, E., MacCor-
mac, L., McGrath, Y., Cleverley, S.,
Lilley, C. E., et al. (2003). Dele-
tion of the virion host shutoff pro-
tein (vhs) from herpes simplex virus
(HSV) relieves the viral block to
dendritic cell activation: potential of
vhs-HSV vectors for dendritic cell-
mediated immunotherapy. J. Virol.
77, 3768–3776.
Shortman, K., and Heath, W. R. (2010).
The CD8+dendritic cell subset.
Immunol. Rev. 234, 18–31.
Sigal, L. J., Crotty, S., Andino, R.,
and Rock, K. L. (1999). Cytotoxic
T-cell immunity to virus-infected
non-haematopoietic cells requires
presentation of exogenous antigen.
Nature 398, 77–80.
Smiley, J. R. (2004). Herpes simplex
virus virion host shutoff protein:
immune evasion mediated by a viral
RNase? J. Virol. 78, 1063–1068.
Smith, C. M., Belz, G. T., Wilson,
N. S., Villadangos, J. A., Short-
man, K., Carbone, F. R., et al.
(2003). Cutting edge: conventional
CD8 alpha+dendritic cells are pref-
erentially involved in CTL priming
after footpad infection with herpes
simplex virus-1. J. Immunol. 170,
4437–4440.
Snyder, C. M., Allan, J. E., Bonnett, E. L.,
Doom, C. M., and Hill,A. B. (2010).
Cross-presentation of a spread-
defective MCMV is sufficient to
prime the majority of virus-specific
CD8+T cells. PLoS ONE 5, e9681.
doi:10.1371/journal.pone.0009681
Song, B., Yeh, K. C., Liu, J., and Knipe,
D. M. (2001). Herpes simplex virus
gene products required for viral
inhibition of expression of G1-phase
functions. Virology 290, 320–328.
Spencer,C. A., Dahmus, M. E., and Rice,
S. A. (1997). Repression of host RNA
polymerase II transcription by her-
pes simplex virus type 1. J. Virol. 71,
2031–2040.
Strelow, L. I., and Leib, D. A. (1996).
Analysis of conserved domains of
UL41 of herpes simplex virus type
1 in virion host shutoff and patho-
genesis. J. Virol. 70, 5665–5667.
Tigges, M. A., Leng, S., Johnson, D. C.,
and Burke, R. L. (1996). Human
herpes simplex virus (HSV)-specific
CD8+CTL clones recognize HSV-
2-infected fibroblasts after treatment
with IFN-gamma or when virion
host shutoff functions are disabled.
J. Immunol. 156, 3901–3910.
Torti, N., Walton, S. M., Murphy, K.
M., and Oxenius, A. (2011). Batf3
transcription factor-dependent DC
subsets in murine CMV infection:
differential impact on T-cell prim-
ing and memory inflation. Eur. J.
Immunol. 41, 2612–2618.
Tortorella, D., Gewurz, B. E., Furman,
M. H., Schust, D. J., and Ploegh,
H. L. (2000). Viral subversion of
the immune system. Annu. Rev.
Immunol. 18, 861–926.
Wagner, M., Gutermann, A., Podlech, J.,
Reddehase, M. J., and Koszinowski,
U. H. (2002). Major histocompati-
bility complex class I allele-specific
www.frontiersin.org November 2012 | Volume 3 | Article 348 | 9

Nopora et al. Cross-priming enhances generation of virus-specific CTL
cooperative and competitive inter-
actions between immune evasion
proteins of cytomegalovirus. J. Exp.
Med. 196, 805–816.
Wilson, N. S., Behrens, G. M., Lundie,
R. J., Smith, C. M., Waithman, J.,
Young, L., et al. (2006). Systemicacti-
vation of dendritic cells by Toll-like
receptor ligands or malaria infec-
tion impairs cross-presentation and
antiviral immunity. Nat.Immunol. 7,
165–172.
Yewdell, J. W., and Hill, A. B. (2002).
Viral interference with antigen
presentation. Nat. Immunol. 3,
1019–1025.
Ziegler,H., Thale, R., Lucin, P.,Muranyi,
W., Flohr, T., Hengel, H., et al.
(1997). A mouse cytomegalovirus
glycoprotein retains MHC class
I complexes in the ERGIC/cis-
Golgi compartments. Immunity 6,
57–66.
Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any
commercial or financial relationships
that could be construed as a potential
conflict of interest.
Received: 24 October 2012; paper pend-
ing published: 31 October 2012; accepted:
05 November 2012; published online: 26
November 2012.
Citation: Nopora K, Bernhard CA, Ried
C, Castello AA, Murphy KM, Mar-
coni P, Koszinowski U and Brocker T
(2012) MHC class I cross-presentation by
dendritic cells counteracts viral immune
evasion. Front. Immun. 3:348. doi:
10.3389/fimmu.2012.00348
This article was submitted to Frontiers
in Antigen Presenting Cell Biology, a
specialty of Frontiers in Immunology.
Copyright © 2012 Nopora, Bernhard,
Ried, Castello, Murphy, Marconi, Koszi-
nowski and Brocker . This is an open-
access article distributed under the terms
of the Creative Commons Attribution
License, which permits use, distribution
and reproduction in other forums, pro-
vided the original authors and source
are credited and subject to any copy-
right notices concerning any third-party
graphics etc.
Frontiers in Immunology | Antigen Presenting Cell Biology November 2012 | Volume 3 | Article 348 | 10