of January 9, 2016.
This information is current as
and Enhances HIV Gag-Specific T Cell
Antigen Processing in Dendritic Cell Subsets
Immunostimulatory Complexes Modifies
Polyinosinic:Polycytidylic Acid and
Johnson, Barbara J. Flynn, Karin Loré and Robert A. Seder
A. Darrah, Ross W. B. Lindsay, Sonia T. Hegde, Teresa R.
Kylie M. Quinn, Ayako Yamamoto, Andreia Costa, Patricia
2013; 191:5085-5096; Prepublished online 2
, 35 of which you can access for free at:
cites 74 articles
is online at:
The Journal of Immunology
Information about subscribing to
Submit copyright permission requests at:
Receive free email-alerts when new articles cite this article. Sign up at:
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
All rights reserved.
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on January 9, 2016
by guest on January 9, 2016
The Journal of Immunology
Coadministration of Polyinosinic:Polycytidylic Acid and
Immunostimulatory Complexes Modifies Antigen Processing
in Dendritic Cell Subsets and Enhances HIV Gag-Specific
T Cell Immunity
Kylie M. Quinn,* Ayako Yamamoto,* Andreia Costa,*,1Patricia A. Darrah,*
Ross W. B. Lindsay,*,2Sonia T. Hegde,*,3Teresa R. Johnson,*,4Barbara J. Flynn,*
Karin Lore ´,*,†and Robert A. Seder*
Currently approved adjuvants induce protective Ab responses but are more limited for generating cellular immunity. In this study,
we assessed the effect of combining two adjuvants with distinct mechanisms of action on their ability to prime T cells: the TLR3
ligand, polyinosinic:polycytidylic acid (poly I:C), and immunostimulatory complexes (ISCOMs). Each adjuvant was administered
alone or together with HIV Gag protein (Gag), and the magnitude, quality, and phenotype of Gag-specific T cell responses were
assessed. For CD8 T cells, all adjuvants induced a comparable response magnitude, but combining poly I:C with ISCOMs induced
a high frequency of CD127+, IL-2–producing cells with decreased expression of Tbet compared with either adjuvant alone. For
CD4 T cells, combining poly I:C and ISCOMs increased the frequency of multifunctional cells, producing IFN-g, IL-2, and TNF,
and the total magnitude of the response compared with either adjuvant alone. CD8 or CD4 T cell responses induced by both
adjuvants mediated protection against Gag-expressing Listeria monocytogenes or vaccinia viral infections. Poly I:C and ISCOMs
can alter Ag uptake and/or processing, and we therefore used fluorescently labeled HIV Gag and DQ-OVA to assess these
mechanisms, respectively, in multiple dendritic cell subsets. Poly I:C promoted uptake and retention of Ag, whereas ISCOMs
enhanced Ag degradation. Combining poly I:C and ISCOMs caused substantial death of dendritic cells but persistence of
degraded Ag. These data illustrate how combining adjuvants, such as poly I:C and ISCOMs, that modulate Ag processing and
have potent innate activity, can enhance the magnitude, quality, and phenotype of T cell immunity.
2013, 191: 5085–5096.
humoral and cellular immune responses can wane following vac-
cination, continued boosting may be required to maintain responses
above a threshold necessary to mediate protection. Protein-based
vaccines given with adjuvants are one approach that can be used in
combination with other vaccine platforms for priming and/or
The Journal of Immunology,
reventive vaccination against HIV, malaria, and tubercu-
losis will require induction of potent Ab responses, T cell
responses, or both for optimal protection (1–4). Because
boosting adaptive immunity. Currently approved clinical adju-
vant formulations include alum and oil/water emulsions, which
elicit protective humoral immunity, but are far less potent for
inducing CD4/Th1 or CD8 T cell immunity [reviewed in (5)]. Im-
proving cellular immunity with protein-based vaccination will re-
quire adjuvants that elicit potent innate cytokines conducive to
induction of cellular responses and efficient Ag presentation.
Polyinosinic:polycytidylic acid (poly I:C) and immunostimula-
tory complexes (ISCOMs) are two adjuvants that show promise in
preclinical studies and early clinical trials for induction of both
Ab and T cell responses (6–9).
Poly I:C is a synthetic dsRNA analog and a ligand for multi-
ple pathogen recognition receptors, including TLR3, melanoma dif-
ferentiation-associated protein 5, retinoic acid-inducible gene 1, and
dsRNA-dependent protein kinase R (10–14). Expression of TLR3
is endosomal and found predominantly in CD8a+dendritic cells
(DCs) or langerin+dermal DCs (dDCs) (15, 16), whereas melanoma
differentiation-associated protein 5, retinoic acid-inducible gene 1,
and protein kinase R localize to the cytosol and are more broadly
expressed on APCs and nonhematopoietic stromal cells (6, 17,
18). Poly I:C stimulates rapid production of IL-6, IL-10, IL-12
p40, MCP-1, TNF, type I IFN, and IFN-g, resulting in significant
DC and NK cell activation (6, 19). When coadministered with
protein Ag, poly I:C potently primes CD4/Th1 cell and Ab responses
(6, 7, 20) and promotes cross-presentation of Ag to CD8 T cells by
DCs through TLR3 signaling (21).
ISCOM particles are cage-like structures that assemble from
*Vaccine Research Center, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD 20892; and†Center for Infectious Med-
icine, Department of Medicine, Karolinska Institutet, Stockholm, SE-171 77, Sweden
1Current address: Fred Hutchinson Cancer Research Center, Seattle, WA.
2Current address: International AIDS Vaccine Initiative, Brooklyn, NY.
3Current address: Centers for Disease Control and Prevention, Atlanta, GA.
4Current address: GenVec, Gaithersburg, MD.
Received for publication June 28, 2013. Accepted for publication September 8, 2013.
This work was supported in part by a grant from the Foundation for the National
Institutes of Health with support from Collaboration for AIDS Vaccine Discovery
Award OPP1039775 from the Bill and Melinda Gates Foundation.
Address correspondence and reprint requests to Dr. Robert A. Seder, Cellular Immu-
nology Section, Vaccine Research Center, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, 40 Convent Drive MSC 3025, Building 40,
Room 3512, Bethesda, MD 20892. E-mail address: email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this article: AF, Alexa Fluor; DC, dendritic cell; dDC, dermal
DC; dLN, draining lymph node; ICS, intracellular cytokine staining; ISCOM, immu-
nostimulatory complex; KLRG1, killer-cell lectin–like receptor subfamily G1; MFI,
median fluorescent intensity; pDC, plasmacytoid DC; poly I:C, polyinosinic:polycy-
tidylic acid; WT, wild-type.
by guest on January 9, 2016
can enhance Ag delivery to APCs when Ag is incorporated into the
particle, but ISCOMs do not function solely as delivery vehicles,
because certain fractions of saponin possess intrinsic adjuvant ac-
tivity (23). ISCOMs have been shown to induce caspase-dependent
cleavage of IL-1b and robust serum production of IL-5, IL-6, GM-
CSF, and IL-12 p40 (24, 25). As a result, ISCOMs prime potent
long-lived Ab responses with a balanced CD4 Th1/Th2 T cell re-
sponse (26) and low-level induction of CTLs. ISCOMs lead to
cross-presentation most likely as a result of disruption of the in-
tegrity of phagolysosomes after endocytosis, which could permit
access of Ag to the cytosol (27, 28). Cross-presentation with
ISCOMs in vitro is most efficient with monocyte-derived DCs (28),
although ex vivo CD8a+DCs are responsible for the majority of Ag
presentation to CD8 T cells (25).
A combination of poly I:C and ISCOMs could potentiate the
effect of each adjuvant by activating distinct, but complimentary
innate signaling and Ag-processing pathways. Prior studies using
combined ligands for distinct TLRs have demonstrated enhanced
innate or adaptive immunity in vitro (29) and in vivo (30). Poly I:C
has been used in combination with particulate delivery systems,
such as liposomes, and combined with other TLR agonists, such as
CpG, to enhance innate signaling and priming of T cells (31, 32).
ISCOMs have also been used in combination with CpG, which
enhanced cross-priming of tumor Ag (33) and induced robust HIV
Env-specific humoral immunity (34). However, a combination of
poly I:C with ISCOMs has not been evaluated.
For this study, we hypothesized that combining poly I:C and
ISCOMs would result in more potent T cell immunity than either
adjuvant alone. We show that combining the adjuvants increased
CD4/Th1 cell responses and enhanced the qualitative and phe-
notypic profile of CD8 T cell responses by increasing expression of
CD127 and IL-2. Combining poly I:C with ISCOMs also resulted
in rapid initial degradation but prolonged retention of Ag, which
may represent a mechanism contributing to the observed effects on
T cells. Overall, the data presented show how combining adjuvants
with distinct effects on innate cytokine production and Ag pre-
sentation can enhance T cell immunity with protein-based vac-
Materials and Methods
BALB/c, C57BL/6, or Batf32/2(C.129S-Batf3tm1Kmm/J) mice were
obtained from The Jackson Laboratory (Bar Harbor, ME) and housed at the
Vaccine Research Center Biomedical Research Unit (Bethesda, MD). Mice
were 6–12 wk old at the time of vaccination. All experimental animal
protocols were approved by the Vaccine Research Center Animal Care and
Formulations and vaccinations
Formulations were prepared using 30 mg HIV-Gag p41 protein (Gag)
(Protein Sciences, Meriden, CT), which was mixed with 12 mg Abisco100
ISCOMs (Isconova AB, Stockholm, Sweden) and/or 50 mg poly I:C
(Invivogen, San Diego, CA) immediately prior to vaccination. In Supple-
mental Fig. 3, 50 mg CpG 1826 (Pfizer, New York, NY) was used. The
ISCOM dose was chosen based on manufacturer’s recommendation, and
the poly I:C, CpG, and protein doses were chosen based on dose titrations
to maximize T cell responses.
Following purification of Gag protein, it was treated with Triton X-100
to remove residual endotoxin and was validated at ,0.1 endotoxin units
with an Endpoint Chromogenic LAL Assay (Lonza, Basel, Swizerland), as
has been performed for prior studies (35, 36). AbISCO100 is a Matrix
formulation that is mixed with Ag in solution and, as such, the Ag is not
incorporated in the structure. All formulations were prepared in PBS and
then administered s.c. in both hind footpads, given as a 100 mL dose split
into 50 mL per footpad. Two doses were administered 3 wk apart.
To assess Ag uptake and degradation experiments, 30 mg HIV Gag Alexa
Fluor (AF) 488 (conjugated by Molecular Probes/Life Technologies, Grand
Island, NY), full-length OVA-AF488 (Molecular Probes), or DQ-OVA
(Molecular Probes) was used.
To assess boosting of primed CD8 T cell responses, 1 3 108particle
units of replication-deficient adenovirus rAd5:Gag was given i.m. as a 100
mL dose into the left gluteal muscle (37).
For assessment of killer-cell lectin–like receptor subfamily G1 (KLRG1)
and CD127 expression, splenocytes were harvested and stained, as previ-
ously described (37), using a PE-labeled H-2Kd tetramer loaded with the
immunodominant HIV Gag peptide (AMQMLKETI) (38). For assessment
of Tbet expression, splenocytes were stained with LIVE/DEAD Fixable
Violet viability dye (Life Technologies), blocked with anti-FcgRIII Ab
(clone 2.4G2; 5 mg/ml; BD Pharmingen), surface stained with PE-labeled
tetramer, anti-CD8 allophycocyanin-Cy7 (clone 53-6.7; BioLegend) and
anti-CD62L PE-Cy7 (clone MEL-14; Abcam), fixed, permeabilized with
Fix/Perm and Perm/Wash Buffers (eBiosciences), and intracellularly
stained with anti-CD3 PerCP-Cy5.5 5 (clone 145-2C11; BD Pharmingen)
and anti–Tbet-AF647 (eBiosciences).
Intracellular cytokine staining
For assessment of Ag-specific cytokine production, splenocytes were har-
vested andrestimulated invitro,aspreviouslydescribed(37),usinga peptide
pool comprising 15-mers spanning HIV Gag p41 (each at 2 mg/ml) or full-
length HIV Gag p41 protein (20 mg/ml). For evaluation of IFN-g, IL-2,
TNF, and IL-10 production, samples were then stained, as previously de-
scribed (37). Alternatively, for evaluation of IL-17 production, samples
were stained, as previously described, except anti–IL-2 FITC (clone JES6-
5H4; BD Pharmingen) and anti–IL-17A PE (clone TC11-18H10; BD
Pharmingen) Abs were substituted for anti–IL-10 AF488 and anti–IL-2 PE
Abs during intracellular staining.
DC preparation and staining
Following vaccination, both popliteal lymph nodes were harvested and
pooled from eight (Gag:AF488 staining) or four (DQ-OVA staining)
individual mice for each formulation. DCs were harvested by enzymatic
digestion and enriched by MACS CD11c positive selection, as previously
described (39). Cells were then stained with LIVE/DEAD Fixable Aqua-
Blue viability dye (Life Technologies) and stained by one of two panels, as
follows: 1) Gag:AF488 staining, surface staining for B220-Cy7PE (clone
RA3-6B2; BD Pharmingen), CD8-allophycocyanin Cy7 (clone 53-6.7;
BioLegend), CD11b-AF700 (clone M1/70; BioLegend), Pan-NK-PacificBlue
(clone DX5; BioLegend), CD19-PacificBlue (clone 6D5; BioLegend),
CD11c-PE (clone HL3; BD Pharmingen), CD103-PerCPCy5.5 (clone
2E7; BioLegend), and DEC205 biotin (clone NLDC-145; Miltenyi
Biotec), followed by streptavidin-TexasRedPE (BD Pharmingen), fol-
lowed by intracellular staining for CD3-PECy5 (clone 145-2C11; BD
Pharmingen) and Langerin-AF647 (clone 929F3.01; Dendritics, Lyon,
France); 2) DQ-OVA staining, surface staining for B220-Cy7PE, CD8-
allophycocyanin Cy7, CD11b-AF700, Pan-NK-PacificBlue and CD19-
PacificBlue, CD11c-QD605 (clone HL3; conjugated in house), followed
by intracellular staining for CD3-allophycocyanin (clone 145-2C11;
BD Pharmingen). DC numbers were back calculated to represent cells
recovered per lymph node.
Multiparameter flow cytometry
Samples were resuspended in 0.5% paraformaldehyde before acquisition
using a modified LSR II flow cytometer (BD Biosciences). Results were
analyzed using FlowJo version 9.3, Pestle version 1.6.2, and SPICE version
5.22 software (M. Roederer, Vaccine Research Center, National Institute
of Allergy and Infectious Diseases, National Institutes of Health). Back-
ground cytokine staining was subtracted, as defined by staining in samples
incubated without peptide or protein.
Infections and Ab-mediated depletions
For infectious challenge, attenuated Listeria monocytogenes (DactA, DintB)
or vaccinia virus (thymidine kinase–deficient Western Reserve strain) was
used, each expressing Gag from HIV-1 strain HXBX or strain IIIB, re-
spectively. Infections were performed, as previously described (37), using
a 2 3 107CFU dose of L. monocytogenes expressing Gag (Listeria:Gag) or
a 6.5 3 106PFU dose of vaccinia virus expressing Gag (rVACV:Gag).
Ab-mediated depletion of CD8 and/or CD4 T cells, after vaccination and
prior to challenge, was performed, as previously described (37). Depleting
Abs were provided by F. Finkelman (University of Cincinnati, Cincinnati,
5086 POLY I:C/ISCOMs ENHANCE Ag PROCESSING AND T CELL RESPONSES
by guest on January 9, 2016
test using SPICE software or a two-tailed Mann–Whitney U test using
To assess the effect of combining poly I:C and ISCOMs on the
generation of T cell responses, we formulated each adjuvant alone
or in combination using HIV-Gag protein (Gag) in BALB/c mice
(Fig. 1A). ISCOMs were used as ISCOM-Matrix formulations,
in which there is no defined physical association between the
ISCOM and the Ag. Two doses of each formulation were ad-
ministered s.c. in both rear footpads, 3 wk apart (Fig. 1A). At
the peak response (10 d after the second dose), the magnitude,
quality, and phenotype of Gag-specific CD8 and CD4 T cell
responses were assessed using multiparameter flow cytometry
after MHC class I tetramer staining and/or intracellular cytokine
staining (ICS) (Fig. 1A).
Poly I:C and/or ISCOMs induce CD8 T cell responses of
similar magnitude but distinct phenotype
The frequency of Gag-specific CD8 T cell responses by tetramer
staining was similar when Gag was given with poly I:C alone,
ISCOMs alone, or poly I:C with ISCOMs (Fig. 1B). We next
assessed the phenotype of tetramer+CD8 T cells using CD127,
the IL-7R a-chain, and KLRG1. Differential expression of these
markers can delineate populations of short-lived effector cells
(40) and memory precursor effector cells (41). In addition, CD127+
KLRG1+CD8 T cells are a population of durable long-term
memory cells induced following viral vaccination or prime-boost
vaccination (37, 42). Formulations that contained ISCOMs induced
a significantly higher proportion (∼70–80%) of CD8 T cells
expressing CD127 with or without KLRG1 (Fig. 1C, black arc)
compared with poly I:C alone (∼50%).
Expression of the transcription factor Tbet is strongly induced
with IL-12 signaling and can alter the differentiation, stability, and
functional capacity of CD8 T cells (40, 43). Tbet expression was
upregulated in Gag-specific CD8 T cells after vaccination with
any of the adjuvants relative to the total CD8 T cell population in
naive mice used as a negative control (Fig. 1D). Poly I:C alone
induced the highest median fluorescence intensity (MFI) for Tbet
expression, followed by ISCOMs alone (Fig. 1D, right panel), but
poly I:C with ISCOMs induced a significantly lower MFI com-
pared with either adjuvant alone (Fig. 1D, right panel). These data
show that combining poly I:C and ISCOMs primes a CD8 T cell
population that is distinct in terms of phenotype and transcription
factor expression from that induced by either adjuvant alone.
Poly I:C and/or ISCOMs induce qualitatively different CD8
T cell responses
Using multiparameter flow cytometry following in vitro restim-
ulation with overlapping Gag peptides, we showed that all adju-
vants induced a comparable frequency of Gag-specific CD8 T cells
as assessed by total cytokine (IFN-g, IL-2, and TNF) production
(Fig. 1E). These data are consistent with the magnitude of Gag-
specific CD8 T cell responses observed by tetramer staining (Fig.
1B). Interestingly, formulations that contained ISCOMs signif-
icantly increased the frequency of IL-2–producing cells relative to
poly I:C alone (Fig. 1F), highlighting differences in the cytokine
profile of Gag-specific CD8 T cells.
To extend these findings, we assessed the quality of Gag-specific
CD8 T cell responses using the relative frequency of cells pro-
ducing any combination of IFN-g, IL-2, and TNF at the single-
cell level (44, 45). Poly I:C alone induced predominantly IFN-g+
TNF+or IFN-g+Gag-specific CD8 T cells (Supplemental Fig. 1A)
and a low frequency (Supplemental Fig. 1A) and proportion (Fig.
1G, 3+, black sector) of multifunctional cells, which can produce
all three cytokines (IFN-g, IL-2, and TNF). These data are con-
sistent with the low frequency of IL-2–producing CD8 T cells
observed in Fig. 1F. ISCOMs alone and poly I:C with ISCOMs
induced a high frequency (Supplemental Fig. 1A) and propor-
tion (Fig. 1G, 3+, black sector) of multifunctional cells. However,
ISCOMs alone also induced a significantly higher frequency (Sup-
plemental Fig. 1A) and proportion (Fig. 1G, black arc) of CD8
T cells that did not express IFN-g, such as IL-2+and IL-2+TNF+
cells, compared with poly I:C alone and poly I:C with ISCOMs.
Taken together, these data illustrate that for CD8 T cells, poly I:C
promotes production of IFN-g, whereas ISCOMs promote pro-
duction of IL-2.
CD8 T cells primed by poly I:C and/or ISCOMs are potently
boosted by rAd5
Given that combining poly I:C and ISCOMs altered the quality
and phenotype of CD8 T cells, we assessed the durability of these
cells and their ability to respond to subsequent stimulation in vivo
following a boost with a viral vector. CD8 T cell responses
were evaluated 6 wk after priming, and the frequency of Gag-
specific CD8 T cells had contracted markedly compared with
the peak response, but responses were still comparable across all
adjuvants (Fig. 1H). To evaluate the ability of primed CD8 T cells
to expand in vivo, mice were boosted with 1 3 108PU rAd5:Gag
4 wk after priming, and responses were assessed 2 wk later. The
frequency of Gag-specific CD8 T cells was significantly higher in
groups that had been primed with any of the adjuvanted vaccines
compared with mice primed with PBS or Gag alone (Fig. 1I).
Additionally, the frequency of Gag-specific CD8 T cells in mice
primed with poly I:C with ISCOMs was significantly higher
compared with poly I:C alone and trended higher compared with
ISCOMs alone (Fig. 1I). Thus, formulations containing ISCOMs
prime CD8 T cell populations that are more responsive to sub-
sequent boosting, and this may be further enhanced with inclusion
of poly I:C.
Combining poly I:C with ISCOMs increases the magnitude of
multifunctional CD4 T cell responses
We next assessed the magnitude and qualitative profile of Gag-
specific CD4 T cell responses. Poly I:C with ISCOMs primed
significantly higher total cytokine responses than either adjuvant
alone (Fig. 2A). ISCOMs alone induced a significantly lower
frequency of IFN-g–producing cells than formulations containing
poly I:C (Fig. 2B). This resulted in marked differences in the
quality of CD4 T cell cytokine responses. Poly I:C alone induced
a high proportion of multifunctional cells (Fig. 2C, 3+, black
sector), whereas ISCOMs alone primed predominantly IL-2+
TNF+, IL-2+or TNF+CD4 T cells (Supplemental Fig. 1B), which
are populations that do not produce IFN-g (Fig. 2C, IFN-g2, black
arc). Poly I:C with ISCOMs induced a high frequency and pro-
portion of IL-2+TNF+and multifunctional CD4 T cells (Sup-
plemental Fig. 1B, Fig. 2C), reflecting the qualitative profiles of
both adjuvants. In general, we observed a clear polarization bias,
in which poly I:C promoted a strong Th1 response with a higher
proportion of IFN-g–producing CD4 T cells compared with
ISCOMs. When we assessed the durability of CD4 T cells,
responses had contracted modestly (1- to 2-fold) but comparably
across adjuvant formulations by 6 wk after priming (Fig. 2D).
The Journal of Immunology 5087
by guest on January 9, 2016
IFN-g, IL-2, and TNF are critical cytokines for Th1 immunity
and protection with a variety of intracellular pathogens. As noted
above, poly I:C potently induced IFN-g production in CD4 T cells,
most likely due to induction of IL-12 and type I and II IFNs (6).
By contrast, CD4 T cell responses induced by ISCOMs were less
polarized toward IFN-g production and Th1 immunity. Given the
heterogeneity of CD4 T cell responses, we assayed for additional
cytokines that may regulate responses or provide alternative ef-
IL-10 produced by CD4 T cells can regulate and promote res-
olution ofimmune responses[reviewed in (46)]. IL-10 was detected
with all adjuvant formulations (Fig. 2E), and the frequencies of IL-
10–producing CD4 T cells were proportionate to the frequencies of
total cytokine response for each adjuvant (Fig. 2A). Both vaccines
(47) and infections (48) can induce CD4 T cells that simulta-
neously induce Th1 cytokine and IL-10 production, and this may
be a mechanism for self-regulation.
ISCOMs induce production of IL-1b (24, 25), which pro-
motes induction of Th17 CD4 T cells in combination with IL-6
(49), and induction of IL-17+CD4 T cells has been observed
after ISCOM vaccination (50). We did not detect IL-17 pro-
duction after vaccination with poly I:C alone, but did detect low
and comparable frequencies of IL-17+CD4 T cells after ISCOM
vaccination, with or without poly I:C in several experiments (Fig.
2F). The biologic importance of such responses remains to be
Protection against Listeria and vaccinia is mediated by CD8
and CD4 T cell responses, respectively
Given the qualitative and quantitative differences in Gag-specific
T cell responses after vaccination, we assessed the ability of these
responses to confer protection against bacterial or viral challenge.
Mice were vaccinated and then challenged 6 wk later with recom-
binant L. monocytogenes or vaccinia virus expressing Gag (Listeria:
Gag and rVACV:Gag, respectively).
After i.v. challenge with Listeria:Gag, spleens were harvested
and bacterial load was evaluated. Mice primed with any of the
adjuvants had a significant but comparable reduction in bacterial
load compared with PBS control or Gag alone (Fig. 3A). After
vaccination with poly I:C with ISCOMs, depletion of CD8 but not
CD4 T cells abrogated protection (Fig. 3B). As all adjuvants in-
duced comparable Gag-specific CD8 T cell responses (Fig. 1B,
1D), these results are consistent with a prior study showing that
the magnitude of CD8 T cell responses correlates with protection
in this model (37).
As an alternative viral challenge model, rVACV:Gag was given
intranasally and body weight was followed over 6 d as a measure
of disease severity. Mice primed with Gag alone or the PBS con-
trol exhibited a marked loss of body weight; however, mice primed
with either poly I:C or ISCOMs alone similarly and significantly
attenuated loss of body weight (Fig. 3C). Strikingly, mice primed
with poly I:C with ISCOMs maintained their original body weight
and were significantly higher than weights observed in mice
primed with poly I:C or ISCOMs alone (Fig. 3C). After vacci-
nation with poly I:C with ISCOMs, depletion of CD4 T cells
with poly I:C and ISCOMs. (A) Experimental schema for this figure and
Fig. 2. BALB/c mice are vaccinated with one of the five indicated com-
binations of Gag (30 mg), poly I:C (50 mg), and ISCOMs (12 mg) given as
two doses, 21 d apart. Splenocytes were harvested 10 d later and used in
MHCI tetramer- or ICS-based assays for T cell responses. (B) Frequency
of CD3+CD8+T cells that are tetramer+. (C) Proportion of CD3+CD8+
tetramer+T cells that express any combination of CD127+or KLRG1+.
The black arc represents the proportion of cells that express CD127. (D)
Histograms (left) and average MFI (right) of CD3+CD8+tetramer+T cells
with each adjuvant as compared with total CD8+T cells from a naive
animal. (E) Frequency of CD3+CD8+T cells producing IFN-g, IL-2, or
TNF (total cytokine+) by ICS. (F) Frequency of CD3+CD8+T cells pro-
ducing IFN-g, IL-2, or TNF individually. (G) Proportion of CD3+CD8+
T cells producing any combination of IFN-g, IL-2, or TNF, in which 3+
cells produce IFN-g, IL-2, and TNF (black), 2+cells produce any two of
IFN-g, IL-2, and TNF (gray), and 1+cells produce IFN-g, IL-2, or TNF
alone (white). The black arc represents cells that do not produce IFN-g.
(H) Frequency of CD3+CD8+T cells producing IFN-g, IL-2, or TNF 6 wk
after the second dose. (I) Frequency of CD3+CD8+T cells producing IFN-g,
Characterization of CD8 T cell responses after vaccination
IL-2, or TNF 2 wk after boosting with rAd5:Gag. Bars and error bars
represent mean 6 SEM. Each group is representative of at least two in-
dependent experiments and four to eight BALB/c mice per group. Statis-
tical differences for bar graphs are represented as *p # 0.05 and **p #
0.01. Statistical differences for pie graphs are represented as#p # 0.05
compared with ISCOMs alone and$p # 0.05 compared with poly I:C
alone. NS, No significant difference.
5088POLY I:C/ISCOMs ENHANCE Ag PROCESSING AND T CELL RESPONSES
by guest on January 9, 2016
caused significantly more weight loss than observed in a control-
treated animal, although not to the same level as the PBS control,
whereas depletion of CD8 T cells caused a mild but not sig-
nificant loss of weight (Fig. 3D). These data suggest that protec-
tion against rVACV:Gag is predominantly dependent on CD4
T cells, with some contribution by CD8 T cells and possibly Ab
responses. This is consistent with the observation that poly I:C with
ISCOMs induced Gag-specific CD4 T cell responses of higher
frequency than poly I:C or ISCOMs alone.
In summary, in these acute infection models, protection corre-
lates directly with magnitude of the relevant T cell response: CD8
for Listeria:Gag and CD4 for rVACV:Gag. It is possible that the
qualitative differences in T cell responses induced by different
adjuvant formulations would have more impact on protection
against more chronic infections.
Poly I:C and ISCOMs differentially alter recruitment of DC
subsets to the draining lymph node
The quantitative and qualitative differences in T cell responses reflect
early activation signals received by T cells from APCs and the innate
environment at the site of priming. Poly I:C is a strong inducer of
typeIIFN, which we havepreviouslyshownto enhancetrafficking
and uptake of Ag by DCs after adjuvanting with a TLR7 ligand
(39). Moreover, ISCOMs disrupt phagolysosomes upon endocy-
tosis by APCs and can alter Ag distribution within Ag-processing
compartments (27, 28). Accordingly, we focused on whether
poly I:C, ISCOMs, or the combination of adjuvants altered Ag
uptake, processing, and presentation by DC subsets that could
subsequently influence priming of T cell immunity.
To assess the kinetics of DC subsets in the draining lymph node
(dLN) after vaccination and their potential for Ag uptake, we admin-
IL-2, or TNF by ICS. (B) Frequency of CD3+CD4+T cells producing IFN-g, IL-2, or TNF individually. (C) Proportion of CD3+CD4+T cells producing any
combination of IFN-g, IL-2, or TNF; 3+, 2+, or 1+cells, and the black arc represents cells that do not produce IFN-g. (D) Frequency of CD3+CD4+T cells
producing IFN-g, IL-2, or TNF 6 wk after the second dose. Frequency of CD3+CD4+T cells producing (E) IL-10 or (F) IL-17 at peak. Bars and error bars
represent mean 6 SEM. Each group is representative of at least two independent experiments and four to eight BALB/c mice per group. Statistical
differences for bar graphs are represented as *p # 0.05. Statistical differences for pie graphs are represented as#p # 0.05 compared with ISCOMs alone and
$p # 0.05 compared with poly I:C alone. NS, No significant difference.
Characterization of CD4 T cell responses after vaccination with poly I:C and ISCOMs. (A) Frequency of CD3+CD4+T cells producing IFN-g,
The Journal of Immunology5089
by guest on January 9, 2016
istered each adjuvant formulation with Gag protein fluorescently la-
processed at 24, 48, 72, and 168 h (1, 2, 3, or 7 d) after vaccination.
Samples were stained with a 12-color multiparameter flow cytometry
panel that enables us to identify six DC subsets present in murine skin
dLNs: the lymph node–resident CD8a+DCs and plasmacytoid DCs
DCs, langerin+and langerin2dDCs, and Langerhans cells (Fig. 4A).
The dLN must be pooled to detect rare DC populations, which limits
statistical analysis between the groups, but the data presented are
representative of three independent experiments with similar results.
After vaccination, there was a gradual increase in the number of
total CD11c+DCs recovered after vaccination with Gag alone, poly I:C
alone, and ISCOMs alone compared with the PBS control or naive
(data not shown) mice at 24 h (Fig. 4B). In contrast, the number of
total CD11c+DCs decreased relative to the PBS control after vac-
cination with poly I:C with ISCOMs at 24 h (Fig. 4B), and there was
a higher number (Fig. 4C) and proportion (Fig. 4C, inset) of dead
leukocytes. This suggests that poly I:C given with ISCOMs induced
death of CD11c+DCs in the dLN.
Upon assessment of DC subset composition in the dLN, we
observed that the number (Fig. 4D, bar graph) and relative fre-
quency (Fig. 4D, pie graph) of pDCs declined markedly by 24 h
after vaccination in all adjuvanted vaccines compared with PBS or
Gag alone. This has been observed after vaccination with other
adjuvants, such as CpG in a protein subunit vaccine (51), and may
represent mobilization to the blood or death of pDCs. Other DC
subsets sequentially increased in number and relative frequency
for all formulations: first, monocyte-derived DCs at 24 h, lan-
gerin+and langerin2dDCs at 48 h, and then Langerhans cells at
72 h (Fig. 4D), reflecting sequential migration of skin-derived DC
subsets to the dLN after vaccination. Of note, vaccination with
ISCOM formulations increases the relative frequency of monocyte-
derived DCs at 48 h, and the number and relative frequency of
monocyte-derived DCs remain elevated at 168 h after vaccination
(Fig. 4D), which was the last time point assessed. This highlights
cination with poly I:C and ISCOMs against
infectious challenge. (A) Bacterial load in
the spleen (CFU) after Listeria:Gag chal-
lenge of mice vaccinated with formulations
containing Gag, ISCOMs, and poly I:C, as
indicated. (B) Bacterial load in the spleen
(CFU) after Listeria:Gag challenge of mice
vaccinated with Gag, ISCOMs, and poly I:C
(vaccine) and either left untreated or treated
with a control Ab (Control Ab), a CD4-de-
pleting Ab (Anti-CD4 Ab), a CD8-depleting
Ab (Anti-CD8 Ab), or both of the latter
(Anti-CD4/8 Abs). (C) Weight loss as per-
centage of original body weight at day 6 after
rVACV:Gag challenge in mice vaccinated
with formulations containing Gag, ISCOMs,
and poly I:C, as indicated. (D) Weight loss as
percentage of original body weight at day 6
after rVACV:Gag challenge in mice vacci-
nated with Gag, ISCOMs, and poly I:C and
left untreated or treated with the indicated
Abs, as above. Bars and error bars represent
geometric mean 6 SE of the geometric mean.
Each group is representative of at least two
independent experiments with four to six
BALB/c mice per group. *p # 0.05, **p #
0.01. NS, No significant difference.
Protection conferred by vac-
5090 POLY I:C/ISCOMs ENHANCE Ag PROCESSING AND T CELL RESPONSES
by guest on January 9, 2016
that ISCOMs induce prolonged recruitment of certain cell subsets,
and monocyte-derived DCs in particular, to the dLN.
Adjuvants modify uptake of Ag by DC subsets following
When uptake of Gag:AF488 was assessed, more Gag:AF488+
CD11c+DCs were recovered after vaccination with poly I:C alone
compared with Gag alone at all time points (Fig. 4E), and Gag:
AF488 was still detected 168 h after vaccination (Fig. 4E, inset).
This is consistent with our previous study in which a TLR7 ligand
induced type I IFN production and thereby increased Ag uptake
(39). In marked contrast, we recovered low numbers of Gag:
AF488+CD11c+DCs from mice that received formulations con-
taining ISCOMs at all time points after vaccination (Fig. 4E).
cytometric plots illustrating gating strategy with CD11c+enriched samples for identification of DC subsets and evaluation of uptake of AF488-labeled Ag. (B)
Number of total live CD11c+DCs recovered per dLN at indicated times after vaccination. (C) Number of total leukocytes in dLN that stain with AquaBlue,
a marker of nonviable cells, and (inset) the proportion of total leukocytes in dLN that are nonviable at 24 h. (D) Number (bar graphs) and relative proportion (pie
graphs) of total live CD11c+DCs that distribute to each DC subset at indicated times after vaccination. (E) Number of live Gag:AF488+CD11c+DCs recovered
per dLN and (inset) the number at 168 h on an expanded y-axis. (F) Number and relative proportion of live Gag:AF488+CD11c+DCs that distribute to each DC
subset at 24 h after vaccination. Results are representative of three independent experiments, with dLN from 10 mice pooled for each formulation.
Composition of DC populations and Ag uptake in dLN of BALB/c mice after vaccination with Gag:AF488, poly I:C, and ISCOMs. (A) Flow
The Journal of Immunology 5091
by guest on January 9, 2016
When DC subsets were assessed for Ag uptake at 24 h after
vaccination, Gag:AF488 localized predominantly to langerin2
dDCs and monocyte-derived DCs for Gag alone or poly I:C alone,
with very low but detectable uptake by CD8a+DCs (Fig. 4F). In
mice that had received ISCOMs with or without poly I:C, there
was a very low number of total Gag:AF488+DCs (Fig. 4F, bar
graphs), but there were higher proportions of Gag:AF488+CD8a+
DCs, Langerhans cells, and langerin+dDCs compared with Gag
alone or poly I:C alone (Fig. 4F, pie graphs). At subsequent time
points, distribution of detectable Ag remained similar to the 24-h
time point for each formulation (data not shown).
These data were collected using BALB/c mice and fluorescently
labeled Gag Ag. Similar results were obtained in C57BL/6 mice
with fluorescently labeled OVA Ag (Supplemental Fig. 2), dem-
onstrating that changes in dLN DC composition and Ag uptake are
not mouse strain or Ag specific.
ISCOMs induce rapid processing of Ag by DCs, but poly I:C
promotes retention of peptide
The limited ability to detect Gag:AF488 in CD11c+DCs after
vaccination with ISCOMs could be due to two distinct mecha-
nisms, as follows: ISCOMs may limit uptake of Ag by DCs or
plots illustrating gating strategy with CD11c+enriched samples for identification of DC subsets and evaluation of degradation of DQ-OVA, indicated by
fluorescence in the B515 channel on a LSRII. (B) Number of live DQ-OVA+CD11c+DCs recovered per dLN at indicated times after vaccination. (C) Dot
plots with frequency of total live CD11c+DCs that are DQ-OVA+. (D) Number and relative proportion of live DQ-OVA+CD11c+DCs that distribute to each
DC subset at indicated times after vaccination. Results are representative of three independent experiments, with dLN from four mice pooled for each
Degradation of Ag by DC subsets in dLN of BALB/c mice after vaccination with DQ-OVA, poly I:C, and ISCOMs. (A) Flow cytometric
5092POLY I:C/ISCOMs ENHANCE Ag PROCESSING AND T CELL RESPONSES
by guest on January 9, 2016
promote rapid degradation of Ag and loss of the AF488 fluores-
cent signal. To examine the rate of degradation of Ag in vivo after
vaccination, we used DQ-OVA. DQ-OVA has high-density BOD-
IPY fluorophore residues arrayed along the OVA protein, which
self-quench when the protein is intact but fluoresce when the
protein undergoes degradation (52, 53).
We simplified the analysis toidentify three dLN DC populations,
as follows: CD8a+DCs, pDCs, and migratory DCs, to include
the monocyte-derived DCs, Langerhans cells, and langerin2and
langerin+dDCs (Fig. 5A). Because Gag:AF488 signal is already
low at 24 h after vaccination with ISCOM formulations (Fig. 4E),
dLNs were harvested from BALB/c mice at 10, 24, and 72 h after
Mice that received ISCOMs alone had a higher number (Fig.
5B) and frequency (Fig. 5C, top and middle rows) of DQ-OVA+
CD11c+DCs at 10 and 24 h after vaccination. Mice that received
poly I:C with ISCOMs had low numbers of DQ-OVA+DCs
compared with ISCOMs alone at 10 and 24 h after vaccination
(Fig. 5B), but both groups had comparable proportions of DCs that
contained DQ-OVA (Fig. 5C, top and middle rows). At early time
points after vaccination with poly I:C with ISCOMs, we observed
significant cell death in the dLN (Fig. 4C), suggesting that the low
number of DQ-OVA+DCs is most likely due to low viability.
Interestingly, we consistently observed an elevated number (Fig.
5B) and frequency (Fig. 5C, bottom row) of DQ-OVA+DCs at 72 h
after vaccination with poly I:C with ISCOMs compared with poly I:C
alone or ISCOMs alone. Finally, degraded OVAwas predominantly
detected in migratory DCs, with very low numbers of DQ-OVA+
CD8a+DCs, but not detected in pDCs (Fig. 5D).
Thus, ISCOMs increase the rate of Ag degradation relative to
poly I:C or protein alone, but inclusion of poly I:C with ISCOMs
promotes persistence of degraded Ag.
Poly I:C and ISCOMs require CD8a+DCs and/or langerin+
dDCs for optimal T cell priming
To identify the DC subsets responsible for priming CD8 and CD4
T cell responses in vivo, we used Batf3-deficient (2/2) mice. Batf3
is a transcription factor essential for development of CD8a+DCs
and langerin+(CD103+) dDCs (54, 55), and, therefore, Batf32/2
mice lack these two DC subsets. CD8a+DCs, langerin+dDCs, and
langerin2dDCs have been shown to mediate cross-presentation
in vivo (39, 56–58) and, as such, would be most likely required
to mediate priming of CD8 T cells with a protein-based vaccine.
Indeed, Gag-specific CD8 T cell cytokine responses were com-
pletely abrogated in Batf32/2mice compared with wild-type
(WT) mice for all adjuvant formulations (Fig. 6A). For CD4
T cells, Batf32/2mice trended toward lower Gag-specific cy-
tokine responses for poly I:C with or without ISCOMs and were
significantly lower for ISCOMs alone compared with WT mice
(Fig. 6B). Overall, CD8a+DCs and langerin+dDCs are essential
for CD8 T cell responses with poly I:C and/or ISCOMs and may
play a role for optimizing CD4 T cell responses with ISCOMs.
Poly I:C and ISCOMs have been used individually in protein-based
vaccines to enhance humoral and cellular immunity in preclinical
and human studies. We focused on poly I:C as an adjuvant based on
recent studies in nonhuman primates, showing that it elicits potent
SIVor HIV Gag T cell responses when given with protein-based or
DC-targeting vaccines (7, 59). Moreover, poly I:C formulated with
poly-lysine and carboxymethylcellulose to enhance stability has
been used as an investigational reagent in humans and shown to
have potent innate activity (60). ISCOM-Matrix vaccines and
vaccines containing the saponin derivative QS21 have been well
tolerated, protective, and generated Ab and T cell responses in
human clinical trials (8, 9, 61). We also noted a significant in-
crease in Gag-specific serum IgG when poly I:C and ISCOMs
were combined, but we focused in this study on quantitative and
qualitative differences in T cell immunity and adjuvant mecha-
nisms that may contribute to these differences, due to the difficulty
in inducing potent T cell responses with protein-based vaccines in
Poly I:C potently induces type I IFN, and coadministration of
poly I:C with Gag in this study led to more DCs loaded with
detectable Ag (Fig. 4D). These data are consistent with our pre-
vious study using a TLR7 ligand adjuvant, which promoted type I
IFN-dependent uptake of Ag by DCs (39). In addition to Ag up-
take, in vitro production of type I IFN by poly I:C promotes re-
tention of degraded Ag by DCs (62). Of note, CpG also induces
robust type I IFN production and can prolong Ag retention and
presentation in vitro (63). Consistent with this in vitro observation,
poly I:C prolonged the presence of degraded Ag in DCs in vivo in
this study (Fig. 5B, 5C), and this correlated with increased CD4
T cell responses after vaccination with the combination of poly I:C
CD3+CD4+T cells producing IFN-g, IL-2, or TNF by ICS after vaccination of WT BALB/c or Batf32/2mice with Gag and each adjuvant formulation.
Bars and error bars represent mean 6 SEM. Each group is representative of at least two independent experiments and four to five mice per group. *p #
0.05. NS, No significant difference.
Role of DC subsets for in vivo priming of T cells after vaccination with poly I:C and/or ISCOMs. Frequency of (A) CD3+CD8+T cells or (B)
The Journal of Immunology5093
by guest on January 9, 2016
and ISCOMs (Fig. 2). Other studies have combined CpG with
ISCOMs and observed increased CD4 T cell immunity (33). As
CpG is also a potent inducer of type I IFN in vivo, we confirmed
that combining CpG with ISCOMs induced quantitative and qual-
itative changes that were similar to poly I:C (Supplemental Fig. 3).
Thus, our results suggest that type I IFN generated by poly I:C, and
possibly CpG, acts on DCs in vivo to prolong retention of degraded
Ag and thereby enhance expansion of Ag-specific CD4 T cell
ISCOMs were shown to accelerate Ag degradation in this study,
which is most likely the result of two possible and not mutually
exclusive mechanisms. Ag codelivered with ISCOMs can enter
the cytosol during disruption, whereupon Ag can be rapidly de-
graded by tripeptidyl peptidase II (28). Alternatively, disruption
of phagolysosomal integrity could trigger cellular death, during
which cellular contents, including Ag, may be degraded, taken
up, and presented by other APCs. When poly I:C and ISCOM
were combined, we observed a high number and frequency of
dead leukocytes soon after vaccination (Fig. 4C). Additionally,
we found that Batf3 expression, and presumably CD8a+DCs
and langerin+dDCs, was required for CD8 T cell responses
(Fig. 6A), despite the fact that these DCs were relatively minor
populations for Ag uptake and degradation (Figs. 4, 5). These
data imply that a very low number of such DCs is required to
directly present Ag or mediate cross-presentation. It was also
notable that CD8a+DCs and langerin+dDCs were required for
optimal CD4 T cell responses after ISCOM vaccination (Fig.
6B). Taken together, we speculate that APCs that initially take
up Ag are not optimal for direct presentation to CD4 T cells,
possibly due to reduced cell viability. Subsequent uptake of
cellular debris with Ag by CD8a+DCs and langerin+dDCs
could facilitate both cross-presentation and MHC class II pre-
Aside from DCs and T cells, other innate cells may shape the
development of adaptive immunity with poly I:C and ISCOMs. In
particular, elevated numbers of neutrophils have been observed in
the dLN after ISCOM vaccination (25), which we also observed
in this study (Supplemental Fig. 4A). Neutrophils may prime or
cross-prime naive TCR transgenic CD4 and CD8 T cells directly
in vivo (64, 65), but neutrophils also undergo cell death in dLNs
(66) and may therefore ferry Ag to secondary lymphoid sites.
We also observed increased and prolonged recruitment of macro-
phages and monocytes (Supplemental Fig. 4B, 4C) and sustained
recruitment of monocyte-derived DCs with the combination of
poly I:C and ISCOMs. Ag presentation by these APC subsets has
been shown to increase T cell responses and promote qualitative
changes such as increased TNF production (67), consistent with
Regardless of the mechanism, the rapid degradation of Ag
would provide an immediate bolus of Ag presentation after
ISCOM vaccination. High Ag density on APCs can engage
T cells with lower-affinity TCRs (68, 69), but subsequent depletion
of Ag can alter CD8 T cell differentiation to increase IL-2 and
TNF production and increase CD127 expression, as observed in
a L. monocytogenes infection model (70). We speculate that this
also occurs after ISCOM vaccination, with rapid presentation of
a high density of Ag that is subsequently depleted, leading to CD8
T cell responses with high IL-2 and TNF production and high
CD127 expression, as observed in this study.
Innate signaling induced by poly I:C or ISCOMs could directly
influence the magnitude of T cell responses. Although not directly
assessed in this work, a number of prior studies have clearly shown
that poly I:C is a potent inducer of type I and II IFNs and IL-12
in vivo (6, 11), which leads to polarized Th1 responses and pro-
motes expansion and survival of CD8 T cells (71–73). Alterna-
tively, ISCOMs have been shown to elicit IL-1b production (24,
25), which can directly signal to CD4 T cells in vitro to augment
proliferation (71). Concurrent treatment of CD4 T cells in vitro
with type I IFN and IL-1ab has been shown to increase cytokine
responses (74); thus, cytokines induced by poly I:C and ISCOMs
may work in concert to enhance the magnitude of CD4/Th1 re-
sponses and the quality of CD8 T cell responses.
In conclusion, the combination of poly I:C and ISCOMs resulted
in protective T cell immunity against bacterial and viral infections.
These data highlight that adjuvants not only have a major role for
enhancing T cell responses through innate cytokines, but also have
profound effects on DC trafficking, Ag uptake, and kinetics of Ag
presentation. We speculate that differences in Ag degradation with
the inclusion of ISCOMs and retention of Ag with the inclusion of
poly I:C may contribute to differences in the magnitude and quality
of T cell responses. As a result, the robust T cell responses induced
by combining poly I:C and ISCOMs are most likely shaped by the
collective effects of Ag presentation and innate signaling. To en-
hance rational design of subunit vaccines, the net effect of multiple
mechanisms altering Ag processing and innate signaling can be
harnessed to augment T cell immunity.
We acknowledge Allison Malloy, Tracy Ruckwardt, and Barney Graham
(Vaccine Research Center) for helpful discussion. We additionally thank
Christine Trumpfheller (The Rockefeller University, New York, NY) for
provision of rVACV:Gag and Fred D. Finkelman (University of Cincinnati,
Cincinnati, OH) for provision of Abs used for depletion studies.
The authors have no financial conflicts of interest.
1. Kwong, P. D., J. R. Mascola, and G. J. Nabel. 2012. The changing face of HIV
vaccine research. J. Int. AIDS Soc. 15: 17407.
2. Hansen, S. G., J. C. Ford, M. S. Lewis, A. B. Ventura, C. M. Hughes, L. Coyne-
Johnson, N. Whizin, K. Oswald, R. Shoemaker, T. Swanson, et al. 2011. Pro-
found early control of highly pathogenic SIV by an effector memory T-cell
vaccine. Nature 473: 523–527.
3. Epstein, J. E., K. Tewari, K. E. Lyke, B. K. L. Sim, P. F. Billingsley,
M. B. Laurens, A. Gunasekera, S. Chakravarty, E. R. James, M. Sedegah, et al.
2011. Live attenuated malaria vaccine designed to protect through hepatic CD8⁺
T cell immunity. Science 334: 475–480.
4. Abel, B., M. Tameris, N. Mansoor, S. Gelderbloem, J. Hughes, D. Abrahams,
L. Makhethe, M. Erasmus, M. de Kock, L. van der Merwe, et al. 2010. The novel
tuberculosis vaccine, AERAS-402, induces robust and polyfunctional CD4+ and
CD8+ T cells in adults. Am. J. Respir. Crit. Care Med. 181: 1407–1417.
5. Coffman, R. L., A. Sher, and R. A. Seder. 2010. Vaccine adjuvants: putting
innate immunity to work. Immunity 33: 492–503.
6. Longhi, M. P., C. Trumpfheller, J. Idoyaga, M. Caskey, I. Matos, C. Kluger,
A. M. Salazar, M. Colonna, and R. M. Steinman. 2009. Dendritic cells require
a systemic type I interferon response to mature and induce CD4+ Th1 immunity
with poly IC as adjuvant. J. Exp. Med. 206: 1589–1602.
7. Flynn, B. J., K. Kastenmu ¨ller, U. Wille-Reece, G. D. Tomaras, M. Alam,
R. W. Lindsay, A. M. Salazar, B. Perdiguero, C. E. Gomez, R. Wagner, et al.
2011. Immunization with HIV Gag targeted to dendritic cells followed by
recombinant New York vaccinia virus induces robust T-cell immunity in non-
human primates. Proc. Natl. Acad. Sci. USA 108: 7131–7136.
8. Drane, D., E. Maraskovsky, R. Gibson, S. Mitchell, M. Barnden, A. Moskwa,
D. Shaw, B. Gervase, S. Coates, M. Houghton, and R. Basser. 2009. Priming of
CD4+ and CD8+ T cell responses using a HCV core ISCOMATRIX vaccine:
a phase I study in healthy volunteers. Hum. Vaccin. 5: 151–157.
9. Pedersen, G. K., A. S. Madhun, L. Breakwell, K. Hoschler, H. Sjursen,
R. D. Pathirana, J. Goudsmit, and R. J. Cox. 2012. T-helper 1 cells elicited by
H5N1 vaccination predict seroprotection. J. Infect. Dis. 206: 158–166.
10. Offermann, M. K., J. Zimring, K. H. Mellits, M. K. Hagan, R. Shaw,
R. M. Medford, M. B. Mathews, S. Goodbourn, and R. Jagus. 1995. Activation
of the double-stranded-RNA-activated protein kinase and induction of vascular
cell adhesion molecule-1 by poly (I).poly (C) in endothelial cells. Eur. J. Bio-
chem. 232: 28–36.
11. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition
of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.
Nature 413: 732–738.
5094 POLY I:C/ISCOMs ENHANCE Ag PROCESSING AND T CELL RESPONSES
by guest on January 9, 2016
12. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi,
M. Miyagishi, K. Taira, S. Akira, and T. Fujita. 2004. The RNA helicase RIG-I
has an essential function in double-stranded RNA-induced innate antiviral
responses. Nat. Immunol. 5: 730–737.
13. Kato, H., O. Takeuchi, E. Mikamo-Satoh, R. Hirai, T. Kawai, K. Matsushita,
A. Hiiragi, T. S. Dermody, T. Fujita, and S. Akira. 2008. Length-dependent
recognition of double-stranded ribonucleic acids by retinoic acid-inducible
gene-I and melanoma differentiation-associated gene 5. J. Exp. Med. 205:
14. Kato, H., O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui,
S. Uematsu, A. Jung, T. Kawai, K. J. Ishii, et al. 2006. Differential roles of
MDA5 and RIG-I helicases in the recognition of RNAviruses. Nature 441: 101–
15. Edwards, A. D., S. S. Diebold, E. M. C. Slack, H. Tomizawa, H. Hemmi,
T. Kaisho, S. Akira, and C. Reis e Sousa. 2003. Toll-like receptor expression in
murine DC subsets: lack of TLR7 expression by CD8 alpha+ DC correlates with
unresponsiveness to imidazoquinolines. Eur. J. Immunol. 33: 827–833.
16. Jelinek, I., J. N. Leonard, G. E. Price, K. N. Brown, A. Meyer-Manlapat,
P. K. Goldsmith, Y. Wang, D. Venzon, S. L. Epstein, and D. M. Segal. 2011.
TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic
cell cross-presentation, CTL responses, and antiviral protection. J. Immunol.
17. Kang, D.-C., R. V. Gopalkrishnan, L. Lin, A. Randolph, K. Valerie, S. Pestka,
and P. B. Fisher. 2004. Expression analysis and genomic characterization of
human melanoma differentiation associated gene-5, mda-5: a novel type I
interferon-responsive apoptosis-inducing gene. Oncogene 23: 1789–1800.
18. Wang, Y., M. Cella, S. Gilfillan, and M. Colonna. 2010. Cutting edge: poly-
inosinic:polycytidylic acid boosts the generation of memory CD8 T cells through
melanoma differentiation-associated protein 5 expressed in stromal cells. J.
Immunol. 184: 2751–2755.
19. Salem, M. L., S. A. El-Naggar, A. Kadima, W. E. Gillanders, and D. J. Cole.
2006. The adjuvant effects of the Toll-like receptor 3 ligand polyinosinic-
cytidylic acid poly (I:C) on antigen-specific CD8+ T cell responses are par-
tially dependent on NK cells with the induction of a beneficial cytokine milieu.
Vaccine 24: 5119–5132.
20. Tewari, K., B. J. Flynn, S. B. Boscardin, K. Kastenmueller, A. M. Salazar,
C. A. Anderson, V. Soundarapandian, A. Ahumada, T. Keler, S. L. Hoffman,
et al. 2010. Poly(I:C) is an effective adjuvant for antibody and multi-functional
CD4+ T cell responses to Plasmodium falciparum circumsporozoite protein
(CSP) and aDEC-CSP in non human primates. Vaccine 28: 7256–7266.
21. Datta, S. K., V. Redecke, K. R. Prilliman, K. Takabayashi, M. Corr, T. Tallant,
J. DiDonato, R. Dziarski, S. Akira, S. P. Schoenberger, and E. Raz. 2003. A
subset of Toll-like receptor ligands induces cross-presentation by bone marrow-
derived dendritic cells. J. Immunol. 170: 4102–4110.
22. Drane, D., C. Gittleson, J. Boyle, and E. Maraskovsky. 2007. ISCOMATRIX
adjuvant for prophylactic and therapeutic vaccines. Expert Rev. Vaccines 6: 761–
23. Behboudi, S., B. Morein, and B. Ro ¨nnberg. 1995. Isolation and quantification of
Quillaja saponaria Molina saponins and lipids in iscom-matrix and iscoms.
Vaccine 13: 1690–1696.
24. Villacres-Eriksson, M., M. Bergstro ¨m-Mollaoglu, H. Ka ˚berg, K. Lo ¨vgren, and
B. Morein. 1993. The induction of cell-associated and secreted IL-1 by iscoms,
matrix or micelles in murine splenic cells. Clin. Exp. Immunol. 93: 120–125.
25. Duewell, P., U. Kisser, K. Heckelsmiller, S. Hoves, P. Stoitzner, S. Koernig,
A. B. Morelli, B. E. Clausen, M. Dauer, A. Eigler, et al. 2011. ISCOMATRIX
adjuvant combines immune activation with antigen delivery to dendritic cells
in vivo leading to effective cross-priming of CD8+ T cells. J. Immunol. 187: 55–
26. Maloy, K. J., A. M. Donachie, and A. M. Mowat. 1995. Induction of Th1 and
Th2 CD4+ T cell responses by oral or parenteral immunization with ISCOMS.
Eur. J. Immunol. 25: 2835–2841.
27. Maraskovsky, E., M. Schnurr, N. S. Wilson, N. C. Robson, J. Boyle, and
D. Drane. 2009. Development of prophylactic and therapeutic vaccines using the
ISCOMATRIX adjuvant. Immunol. Cell Biol. 87: 371–376.
28. Schnurr, M., M. Orban, N. C. Robson, A. Shin, H. Braley, D. Airey, J. Cebon,
E. Maraskovsky, and S. Endres. 2009. ISCOMATRIX adjuvant induces efficient
cross-presentation of tumor antigen by dendritic cells via rapid cytosolic antigen
delivery and processing via tripeptidyl peptidase II. J. Immunol. 182: 1253–
29. Napolitani, G., A. Rinaldi, F. Bertoni, F. Sallusto, and A. Lanzavecchia. 2005.
Selected Toll-like receptor agonist combinations synergistically trigger a T
helper type 1-polarizing program in dendritic cells. Nat. Immunol. 6: 769–776.
30. Kasturi, S. P., I. Skountzou, R. A. Albrecht, D. Koutsonanos, T. Hua, H. I. Nakaya,
R. Ravindran, S. Stewart, M. Alam, M. Kwissa, et al. 2011. Programming the
magnitude and persistence of antibody responses with innate immunity. Nature 470:
31. Nordly, P., F. Rose, D. Christensen, H. M. Nielsen, P. Andersen, E. M. Agger,
and C. Foged. 2011. Immunity by formulation design: induction of high CD8+
T-cell responses by poly(I:C) incorporated into the CAF01 adjuvant via a double
emulsion method. J. Control. Release 150: 307–317.
32. Zaks, K., M. Jordan, A. Guth, K. Sellins, R. Kedl, A. Izzo, C. Bosio, and S. Dow.
2006. Efficient immunization and cross-priming by vaccine adjuvants containing
TLR3 or TLR9 agonists complexed to cationic liposomes. J. Immunol. 176:
33. Jacobs, C., P. Duewell, K. Heckelsmiller, J. Wei, F. Bauernfeind, J. Ellermeier,
U. Kisser, C. A. Bauer, M. Dauer, A. Eigler, et al. 2011. An ISCOM vaccine
combined with a TLR9 agonist breaks immune evasion mediated by regulatory
T cells in an orthotopic model of pancreatic carcinoma. Int. J. Cancer 128: 897–
34. Sundling, C., M. N. E. Forsell, S. O’Dell, Y. Feng, B. Chakrabarti, S. S. Rao,
K. Lore ´, J. R. Mascola, R. T. Wyatt, I. Douagi, and G. B. Karlsson Hedestam.
2010. Soluble HIV-1 Env trimers in adjuvant elicit potent and diverse functional
B cell responses in primates. J. Exp. Med. 207: 2003–2017.
35. Wille-Reece, U., B. J. Flynn, K. Lore ´, R. A. Koup, R. M. Kedl, J. J. Mattapallil,
W. R. Weiss, M. Roederer, and R. A. Seder. 2005. HIV Gag protein conjugated
to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1
and CD8+ T cell responses in nonhuman primates. Proc. Natl. Acad. Sci. USA
36. Wille-Reece, U., B. J. Flynn, K. Lore ´, R. A. Koup, A. P. Miles, A. Saul,
R. M. Kedl, J. J. Mattapallil, W. R. Weiss, M. Roederer, and R. A. Seder. 2006.
Toll-like receptor agonists influence the magnitude and quality of memory T cell
responses after prime-boost immunization in nonhuman primates. J. Exp. Med.
37. Quinn, K. M., A. Da Costa, A. Yamamoto, D. Berry, R. W. B. Lindsay,
P. A. Darrah, L. Wang, C. Cheng, W.-P. Kong, J. G. D. Gall, et al. 2013.
Comparative analysis of the magnitude, quality, phenotype, and protective ca-
pacity of simian immunodeficiency virus gag-specific CD8+ T cells following
human-, simian-, and chimpanzee-derived recombinant adenoviral vector im-
munization. J. Immunol. 190: 2720–2735.
38. Mata, M., P. J. Travers, Q. Liu, F. R. Frankel, and Y. Paterson. 1998. The MHC
class I-restricted immune response to HIV-gag in BALB/c mice selects a single
epitope that does not have a predictable MHC-binding motif and binds to Kd
through interactions between a glutamine at P3 and pocket D. J. Immunol. 161:
39. Kastenmu ¨ller, K., U. Wille-Reece, R. W. B. Lindsay, L. R. Trager, P. A. Darrah,
B. J. Flynn, M. R. Becker, M. C. Udey, B. E. Clausen, B. Z. Igyarto, et al. 2011.
Protective T cell immunity in mice following protein-TLR7/8 agonist-conjugate
immunization requires aggregation, type I IFN, and multiple DC subsets. J. Clin.
Invest. 121: 1782–1796.
40. Joshi, N. S., W. Cui, A. Chandele, H. K. Lee, D. R. Urso, J. Hagman, L. Gapin,
and S. M. Kaech. 2007. Inflammation directs memory precursor and short-lived
effector CD8(+) T cell fates via the graded expression of T-bet transcription
factor. Immunity 27: 281–295.
41. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, and
R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies
effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4:
42. Obar, J. J., E. R. Jellison, B. S. Sheridan, D. A. Blair, Q.-M. Pham,
J. M. Zickovich, and L. Lefranc ¸ois. 2011. Pathogen-induced inflammatory en-
vironment controls effector and memory CD8+ T cell differentiation. J. Immu-
nol. 187: 4967–4978.
43. Intlekofer, A. M., N. Takemoto, E. J. Wherry, S. A. Longworth, J. T. Northrup,
V. R. Palanivel, A. C. Mullen, C. R. Gasink, S. M. Kaech, J. D. Miller, et al.
2005. Effector and memory CD8+ T cell fate coupled by T-bet and eomeso-
dermin. Nat. Immunol. 6: 1236–1244.
44. Darrah, P. A., D. T. Patel, P. M. De Luca, R. W. B. Lindsay, D. F. Davey,
B. J. Flynn, S. T. Hoff, P. Andersen, S. G. Reed, S. L. Morris, et al. 2007.
Multifunctional TH1 cells define a correlate of vaccine-mediated protection
against Leishmania major. Nat. Med. 13: 843–850.
45. Betts, M. R., M. C. Nason, S. M. West, S. C. De Rosa, S. A. Migueles,
J. Abraham, M. M. Lederman, J. M. Benito, P. A. Goepfert, M. Connors, et al.
2006. HIV nonprogressors preferentially maintain highly functional HIV-specific
CD8+ T cells. Blood 107: 4781–4789.
46. Murray, P. J., and S. T. Smale. 2012. Restraint of inflammatory signaling by in-
terdependent strata of negative regulatory pathways. Nat. Immunol. 13: 916–924.
47. Darrah, P. A., S. T. Hegde, D. T. Patel, R. W. B. Lindsay, L. Chen, M. Roederer,
and R. A. Seder. 2010. IL-10 production differentially influences the magnitude,
quality, and protective capacity of Th1 responses depending on the vaccine
platform. J. Exp. Med. 207: 1421–1433.
48. Jankovic, D., D. G. Kugler, and A. Sher. 2010. IL-10 production by CD4+ ef-
fector T cells: a mechanism for self-regulation. Mucosal Immunol. 3: 239–246.
49. Ghoreschi, K., A. Laurence, X.-P. Yang, C. M. Tato, M. J. McGeachy,
J. E. Konkel, H. L. Ramos, L. Wei, T. S. Davidson, N. Bouladoux, et al. 2010.
Generation of pathogenic T(H)17 cells in the absence of TGF-b signalling.
Nature 467: 967–971.
50. Yu, H., X. Jiang, C. Shen, K. P. Karunakaran, J. Jiang, N. L. Rosin, and
R. C. Brunham. 2010. Chlamydia muridarum T-cell antigens formulated with
the adjuvant DDA/TDB induce immunity against infection that correlates with
a high frequency of gamma interferon (IFN-gamma)/tumor necrosis factor alpha
and IFN-gamma/interleukin-17 double-positive CD4+ T cells. Infect. Immun. 78:
51. Shah, J. A., P. A. Darrah, D. R. Ambrozak, T. N. Turon, S. Mendez, J. Kirman,
C.-Y. Wu, N. Glaichenhaus, and R. A. Seder. 2003. Dendritic cells are respon-
sible for the capacity of CpG oligodeoxynucleotides to act as an adjuvant for
protective vaccine immunity against Leishmania major in mice. J. Exp. Med.
52. Santambrogio, L., A. K. Sato, G. J. Carven, S. L. Belyanskaya, J. L. Strominger,
and L. J. Stern. 1999. Extracellular antigen processing and presentation by im-
mature dendritic cells. Proc. Natl. Acad. Sci. USA 96: 15056–15061.
53. Daro, E., B. Pulendran, K. Brasel, M. Teepe, D. Pettit, D. H. Lynch, D. Vremec,
L. Robb, K. Shortman, H. J. McKenna, et al. 2000. Polyethylene glycol-modified
GM-CSF expands CD11b(high)CD11c(high) but notCD11b(low)CD11c(high)
murine dendritic cells in vivo: a comparative analysis with Flt3 ligand. J.
Immunol. 165: 49–58.
The Journal of Immunology5095
by guest on January 9, 2016
54. Hildner, K., B. T. Edelson, W. E. Purtha, M. Diamond, H. Matsushita, Download full-text
M. Kohyama, B. Calderon, B. U. Schraml, E. R. Unanue, M. S. Diamond, et al.
2008. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in
cytotoxic T cell immunity. Science 322: 1097–1100.
55. Edelson, B. T., W. Kc, R. Juang, M. Kohyama, L. A. Benoit, P. A. Klekotka,
C. Moon, J. C. Albring, W. Ise, D. G. Michael, et al. 2010. Peripheral CD103+
dendritic cells form a unified subset developmentally related to CD8alpha+
conventional dendritic cells. J. Exp. Med. 207: 823–836.
56. Allan, R. S., J. Waithman, S. Bedoui, C. M. Jones, J. A. Villadangos, Y. Zhan,
A. M. Lew, K. Shortman, W. R. Heath, and F. R. Carbone. 2006. Migratory
dendritic cells transfer antigen to a lymph node-resident dendritic cell population
for efficient CTL priming. Immunity 25: 153–162.
57. Bedoui, S., P. G. Whitney, J. Waithman, L. Eidsmo, L. Wakim, I. Caminschi,
R. S. Allan, M. Wojtasiak, K. Shortman, F. R. Carbone, et al. 2009. Cross-
presentation of viral and self antigens by skin-derived CD103+ dendritic cells.
Nat. Immunol. 10: 488–495.
58. Henri, S., L. F. Poulin, S. Tamoutounour, L. Ardouin, M. Guilliams, B. de Bovis,
E. Devilard, C. Viret, H. Azukizawa, A. Kissenpfennig, and B. Malissen. 2010.
CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived anti-
gens irrespective of the presence of Langerhans cells. J. Exp. Med. 207: 189–206.
59. Park, H., L. Adamson, T. Ha, K. Mullen, S. I. Hagen, A. Nogueron, A. W. Sylwester,
M. K. Axthelm, A. Legasse, M. Piatak, Jr., et al. 2013. Polyinosinic-polycytidylic
acid is the most effective TLR adjuvant for SIV Gag protein-induced T cell
responses in nonhuman primates. J. Immunol. 190: 4103–4115.
60. Caskey, M., F. Lefebvre, A. Filali-Mouhim, M. J. Cameron, J.-P. Goulet,
E. K. Haddad, G. Breton, C. Trumpfheller, S. Pollak, I. Shimeliovich, et al. 2011.
Synthetic double-stranded RNA induces innate immune responses similar to
a live viral vaccine in humans. J. Exp. Med. 208: 2357–2366.
61. The RTS,S Clinical Trials Partnership. 2012. A phase 3 trial of RTS,S/AS01
malaria vaccine in African infants. N. Engl. J. Med. 367: 2284-2295.
62. Spadaro, F., C. Lapenta, S. Donati, L. Abalsamo, V. Barnaba, F. Belardelli,
S. M. Santini, and M. Ferrantini. 2012. IFN-a enhances cross-presentation in
human dendritic cells by modulating antigen survival, endocytic routing, and
processing. Blood 119: 1407–1417.
63. Kuchtey, J., P. J. Chefalo, R. C. Gray, L. Ramachandra, and C. V. Harding. 2005.
Enhancement of dendritic cell antigen cross-presentation by CpG DNA involves
type I IFN and stabilization of class I MHC mRNA. J. Immunol. 175: 2244–
64. Culshaw, S., O. R. Millington, J. M. Brewer, and I. B. McInnes. 2008. Murine
neutrophils present class II restricted antigen. Immunol. Lett. 118: 49–54.
65. Beauvillain, C., Y. Delneste, M. Scotet, A. Peres, H. Gascan, P. Guermonprez,
V. Barnaba, and P. Jeannin. 2007. Neutrophils efficiently cross-prime naive
T cells in vivo. Blood 110: 2965–2973.
66. Abadie, V., E. Badell, P. Douillard, D. Ensergueix, P. J. M. Leenen, M. Tanguy,
L. Fiette, S. Saeland, B. Gicquel, and N. Winter. 2005. Neutrophils rapidly
migrate via lymphatics after Mycobacterium bovis BCG intradermal vaccination
and shuttle live bacilli to the draining lymph nodes. Blood 106: 1843–1850.
67. Abadie, V., O. Bonduelle, D. Duffy, C. Parizot, B. Verrier, and B. Combadie `re.
2009. Original encounter with antigen determines antigen-presenting cell im-
printing of the quality of the immune response in mice. PLoS One 4: e8159.
68. Mempel, T. R., S. E. Henrickson, and U. H. Von Andrian. 2004. T-cell priming
by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427:
69. Henrickson, S. E., T. R. Mempel, I. B. Mazo, B. Liu, M. N. Artyomov, H. Zheng,
A. Peixoto, M. P. Flynn, B. Senman, T. Junt, et al. 2008. T cell sensing of antigen
dose governs interactive behavior with dendritic cells and sets a threshold for
T cell activation. Nat. Immunol. 9: 282–291.
70. Badovinac, V. P., and J. T. Harty. 2007. Manipulating the rate of memory CD8+
T cell generation after acute infection. J. Immunol. 179: 53–63.
71. Curtsinger, J. M., C. S. Schmidt, A. Mondino, D. C. Lins, R. M. Kedl,
M. K. Jenkins, and M. F. Mescher. 1999. Inflammatory cytokines provide a third
signal for activation of naive CD4+ and CD8+ T cells. J. Immunol. 162: 3256–
72. Zhang, X., S. Sun, I. Hwang, D. F. Tough, and J. Sprent. 1998. Potent and se-
lective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Im-
munity 8: 591–599.
73. Tough, D. F., X. Zhang, and J. Sprent. 2001. An IFN-gamma-dependent pathway
controls stimulation of memory phenotype CD8+ T cell turnover in vivo by IL-
12, IL-18, and IFN-gamma. J. Immunol. 166: 6007–6011.
74. Madera, R. F., J. P. Wang, and D. H. Libraty. 2011. The combination of early and
rapid type I IFN, IL-1a, and IL-1b production are essential mediators of RNA-
like adjuvant driven CD4+ Th1 responses. PLoS One 6: e29412.
5096 POLY I:C/ISCOMs ENHANCE Ag PROCESSING AND T CELL RESPONSES
by guest on January 9, 2016