Privileged Antigen Presentation in Splenic B
Cell Follicles Maximizes T Cell Responses in
Prime-Boost Vaccination
This information is current as
of June 16, 2017.
Byram W. Bridle, Andrew Nguyen, Omar Salem, Liang
Zhang, Sandeep Koshy, Derek Clouthier, Lan Chen,
Jonathan Pol, Stephanie L. Swift, Dawn M. E. Bowdish,
Brian D. Lichty, Jonathan L. Bramson and Yonghong Wan
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J Immunol 2016; 196:4587-4595; Prepublished online 27
April 2016;
doi: 10.4049/jimmunol.1600106
http://www.jimmunol.org/content/196/11/4587
The Journal of Immunology
Privileged Antigen Presentation in Splenic B Cell Follicles
Maximizes T Cell Responses in Prime-Boost Vaccination
Byram W. Bridle,* Andrew Nguyen,† Omar Salem,† Liang Zhang,† Sandeep Koshy,†
Derek Clouthier,† Lan Chen,† Jonathan Pol,† Stephanie L. Swift,† Dawn M. E. Bowdish,†
Brian D. Lichty,† Jonathan L. Bramson,† and Yonghong Wan†
he CD8+ T cell subset plays an essential role in host defense against viruses, intracellular bacteria, and malignancies. Generation of protective CD8+ T cell immunity
has been a central focus for the development of Ag-specific vaccines. Although numerous strategies have been designed to elicit
high frequencies of circulating Ag-specific CD8+ T cells, sequential immunizations with viral vectors (conventionally known
as prime-boost immunization) is one of the most effective (1). The
acute phase of the primary response is dominated by highly differentiated CD8+ effector T cells (TEFF) that display robust cytotoxicity and production of inflammatory cytokines, necessary
for initial control of fatal infections and other severe diseases. However, TEFF have a limited capacity to proliferate and booster immunizations need to optimally stimulate memory T cells, especially
central memory T cells (TCM), which proliferate rapidly and vigorously upon encounter with Ags (2). Although a small number of TCM
T
*Department of Pathobiology, Ontario Veterinary College, University of Guelph,
Guelph, Ontario N1G 2W1, Canada; and †Department of Pathology and Molecular
Medicine, McMaster Immunology Research Centre, McMaster University, Hamilton,
Ontario L8N 3Z5, Canada
ORCIDs: 0000-0002-8335-1118 (B.W.B.); 0000-0002-2512-2384 (L.Z.); 0000-00032836-5813 (S.K.); 0000-0002-8355-7562 (J.P.); 0000-0002-8545-8322 (S.L.S.).
Received for publication January 19, 2016. Accepted for publication April 6, 2016.
This work was supported by grants from the Canadian Institutes of Health Research
and the Ontario Cancer Research Network (to Y.W.).
Address correspondence and reprint requests to Dr. Yonghong Wan, Department
of Pathology and Molecular Medicine, McMaster University, Room MDCL5024, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: Ad, recombinant human adenovirus; DC, dendritic
cell; DCT, dopachrome tautomerase; DT, diphtheria toxin; DTR, diphtheria toxin
receptor; hDCT, human dopachrome tautomerase; LCMV, lymphocytic choriomeningitis virus; Luc, luciferase; SIIN, SIINFEKL; TCM, central memory T cell;
TEFF, effector T cell; TEM, effector memory T cell; VSV, vesicular stomatitis virus;
VV, vaccinia virus; WT, wild-type.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600106
can be identified during the acute phase of a typical primary CD8+
T cell response, subsequent boosting is limited by pre-existing TEFF
that rapidly clear Ag-loaded cells, preventing adequate Ag presentation to TCM (3–5). Thus, prime-boost immunizations in a prophylactic
setting are typically spread across a period of weeks to months to
allow time for the TCM to dominate the available T cell pool and
enable maximal secondary expansion. In the case of therapeutic vaccination for patients with lethal (e.g., Ebola) or chronic infection (e.g.,
HIV, hepatitis C virus) or cancer, where immediate and high levels of
the TEFF are needed for protection, boosting strategies that can bypass TEFF-mediated negative feedback regulation would be desirable.
It is established that activation of naive T cells requires a
coordinated process between migratory dendritic cells (DCs) from
peripheral tissues and lymphoid-resident APCs (6–8), but little is
known about the role of different APCs in Ag presentation to
memory T cells. It was originally thought that memory T cells had a
reduced threshold for activation and, thus, could be triggered to
proliferate by both professional and nonprofessional APCs (9, 10).
Recent work, however, has changed that perspective, as hematopoietic APCs have been shown to be required for secondary expansion (11); one study has suggested that DCs, in particular, may
be required for maximal secondary responses (12). However, Agcarrying DCs are sensitive to TEFF-mediated killing and they can be
rapidly eliminated prior to their engagement with memory cells in
the secondary lymphoid organs (13–15).
Interestingly, we have recently demonstrated that vaccination with
recombinant rhabdoviruses, such as vesicular stomatitis virus (VSV)
and Maraba virus, could provoke massive secondary expansion of
T cells as early as 4 d after priming or at the height of the primary
CD8+ T cell response (7–14 d after priming) (16–18), suggesting
that rhabdoviral boosting can overcome the TEFF barrier, allowing
Ag-loaded APCs to escape TEFF-mediated killing. Understanding
the mechanisms of Ag presentation that facilitate rapid boosting by
rhabdoviruses will provide important information that can be used
to design optimal vaccination strategies for rapid generation of
protective responses.
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Effector T cells (TEFF) are a barrier to booster vaccination because they can rapidly kill Ag-bearing APCs before memory T cells
are engaged. We report in this study that i.v. delivery of rhabdoviral vectors leads to direct infection of follicular B cells in the
spleen, where the earliest evidence of secondary T cell responses was observed. This allows booster immunizations to rapidly
expand CD8+ central memory T cells (TCM) during the acute phase of the primary response that is dominated by TEFF. Interestingly, although the ablation of B cells before boosting with rhabdoviral vectors diminishes the expansion of memory T cells,
B cells do not present Ags directly. Instead, depletion of CD11c+ dendritic cells abrogates secondary T cell expansion, suggesting
that virus-infected follicular B cells may function as an Ag source for local DCs to subsequently capture and present the Ag.
Because TCM are located within B cell follicles in the spleen whereas TEFF cannot traffic through follicular regions, Ag production
and presentation by follicular APCs represent a unique mechanism to secure engagement of TCM during an ongoing effector
response. Our data offer insights into novel strategies for rapid expansion of CD8+ T cells using prime-boost vaccines by targeting
privileged sites for Ag presentation. The Journal of Immunology, 2016, 196: 4587–4595.
4588
In the present study, we demonstrate that a robust secondary CD8+
T cell expansion following booster immunization with VSV occurs
primarily in the spleen. Inducing maximal secondary CD8+ T cell
responses at the peak of the primary response requires i.v. delivery
of VSV, which results in direct infection of follicular B cells and
subsequent Ag presentation by neighboring DCs in the follicular
region. Escape of Ag-loaded APCs from TEFF-mediated killing is
consistent with evidence that TCM are located within B cell follicles
in the spleen whereas TEFF cannot traffic through follicular regions
(19–21). These data reveal a distinct role of splenic APCs with
regard to secondary CD8+ T cell activation, which may not only
offer insights into engineering prime-boost vaccines for rapid expansion of CD8+ T cells but may also provide new fundamental
mechanistic insight into the immune response to viremia.
Materials and Methods
Mice
Viral vectors
The recombinant human adenovirus (Ad)-BHG was a replication-deficient
recombinant human serotype 5 Ad with no transgene inserted and served
as a control vector. The Ad–human dopachrome tautomerase (hDCT)
expressed the full-length hDCT gene. The Ad-SIINFEKL-luciferase (Luc)
(Ad-SIIN) vector expressed the immunodominant epitope of chicken OVA
(OVA257–264) coupled to firefly luciferase. The Ad-gp33 expressed the
immunodominant eptiope from the gp33 of lymphocytic choriomeningitis
virus (LCMV). Recombinant VSV-hDCT and VSV-GFP have been described (22). VSV-SIIN (expressing OVA257–264 and luciferase) and VSVgp33 (expressing gp33–41 from LCMV) were engineered by an identical
construction strategy. The VSV-MT was a control vector lacking a transgene. Recombinant vaccinia virus (VV, Western Reserve strain) expressing
SIINFEKL-Luc (VV-SIINFEKL-Luc) has been described (23). Recombinant Maraba virus expressing hDCT (Maraba-hDCT) was constructed
based on the attenuated strain MG1 of Maraba virus (24).
Peptides
Kb-restricted DCT (DCT180–188, SVYDFFVWL) and OVA (OVA257–264,
SIINFEKL) peptides were synthesized by Pepscan Systems (Lelystad, the
Netherlands). Using a peptide library of 15-mers spanning the full length
of the DCT protein (Pepscan Systems) we identified several CD8+ T cell
epitope–containing peptides, which were pooled for evaluating immune
responses in BALB/c mice.
Abs/tetramers
Abs and kits used for surface or intracellular staining were purchased from
BD Biosciences (Mississauga, ON, Canada). The tetramers Kb-DCT180–188allophycocyanin, Kb-OVA257–264-allophycocyanin, and Db-gp33–41-allophycocyanin were obtained from Baylor College of Medicine.
BrdU incorporation assay
Immunized mice received i.p. injections of 1 mg BrdU and BrdU-containing
drinking water (0.8 mg/ml) 24 h prior to harvesting tissues. To block T cell
trafficking from lymphoid organs in some experiments, mice also received
an i.p. injection of FTY720 (Cayman Chemical, Ann Arbor, Michigan) at
4 mg/kg body weight 24 h before boosting with VSV. After blocking Fc
receptors, lymphocytes in single-cell suspensions from various tissues were
stained with a tetramer and then Abs against surface CD8 and intranuclear
BrdU (BD Biosciences).
CD127+CD62L2), and TCM (CD127+CD62L+) populations. Sorted subsets
(.90% purity) were adoptively transferred into naive wild-type (WT)
C57BL/6 recipients (Thy1.2+) 1 d prior to vaccination with VSV-SIIN.
Five days after administration of VSV, blood, bone marrow, and spleens
were harvested to determine the frequency of congenic tetramer+ cells.
Identification of VSV-infected cells
C57BL/6 mice received 1 3 109 PFU VSV-GFP i.v. Spleens were harvested 1.5 h later to minimize the potential for the confounding effect of
cell trafficking between tissues, while providing enough time for VSV to
infect cells. Splenocytes were cultured for an additional 4.5 h to allow GFP
to accumulate within infected cells for detection by flow cytometry.
B cell depletion
For depletion of B cells, mice were given 250 mg anti-CD20 mAb (clone
18B12, Biogen Idec, San Diego, CA) by the i.v. route 7 d prior to vaccination and 2 wk later to maintain depletion. By flow cytometry, .98% of
B cells were removed from the blood and spleen. Sham-treated mice received an isotype control.
Chimeras
C57BL/6 recipient mice were irradiated (2 3 550 rads; 48-h interval) and
then received i.v. injections of T cell–depleted bone marrow–derived cells.
Four sets of chimeric mice were made: group 1 received 5 3 106 WT cells;
group 2 received 4 3 106 cells from B2/2 mice and 2 3 106 cells from WT
mice; group 3 received 4 3 106 cells from B2/2 mice and 2 3 106 cells
from KbDb2/2 mice; and group 4 received 5 3 106 cells from B2/2 donors.
CD11c-DTR chimeric mice were similarly prepared by transferring CD11cDTR transgenic mouse-derived bone marrow into lethally irradiated C57BL/6
recipients. Mice were given 3 mo to reconstitute their hematopoietic systems, which was confirmed by flow cytometry prior to experimentation.
In vitro coculture of splenic B cells, Ag-specific T cells, and DCs
C57BL/6 or KbDb2/2 mice were injected i.v. with 1 3 109 VSV-GFP or
VSV-gp33. Seventeen hours later, splenic B cells were purified using
negative selection kits according to the manufacturer’s instructions (Miltenyi Biotec, Teterow, Germany). Naive P14 cells were enriched by positive selection from the spleens of P14 mice (EasySep mouse CD8a
positive selection kit, Stemcell Technologies) and labeled with 1 mM
CFSE (Sigma-Aldrich, Oakville, ON, Canada). Bone marrow–derived DCs
were generated and matured as described previously (25).
Purified WT B cells (2 3 107) were cocultured with 2 3 106 bone
marrow–derived DCs for 18 h at 37˚C. DCs were separated using CD11c
microbeads (Miltenyi Biotec) and cultured with CFSE-labeled P14 cells at
a 1:1 ratio. Cultures were analyzed for proliferation after 72 h. Additionally, 5 3 106 KbDb2/2 B cells were directly mixed with 5 3 105
CFSE-labeled P14 cells in a 24-well plate or separated by Transwell inserts
(Corning, Tewksbury, MA) in the presence or absence of 5 3 105 DCs for
72 h prior to flow cytometric analysis.
Splenectomy
Mice primed with Ad-hDCT received splenectomies or sham surgeries 12 d
later. At 48 h after surgery, mice were boosted with 1 3 109 PFU i.v. VSVhDCT. Five days after VSV, DCT180–188-specific CD8+ T cell responses
were quantified by flow cytometric analysis of intracellular cytokine
staining following in vitro peptide restimulation.
Statistical analyses
GraphPad Prism for Windows 6.0 (GraphPad Software, San Diego, CA) was
used for graphing and statistical analyses. When required, data were normalized by log transformation. A Student two-tailed t test, Mann–Whitney
U test, or one- or two-way ANOVA was used to query immune response
data. Differences between means were considered significant at p # 0.05.
Means with SE bars are shown. Survival data were analyzed using the
Kaplan–Meier method and the log-rank test.
Results
Adoptive T cell transfer
Intravenous injection of VSV at the peak of the primary
response rapidly boosts systemic CD8+ T cells to massive
numbers
Negatively selected splenic CD8+ T cells (Stemcell Technologies, Vancouver, BC, Canada) from Ad-SIIN–immunized congenic (Thy1.1+) mice
were sorted into TEFF (CD1272CD62L2), effector memory T cell (TEM,
The choice of i.v. administration of rhabdoviruses (i.e., VSV and
Maraba) in our previous studies was driven by our desire to exploit
their oncolytic potential to target both primary and metastatic tumors
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C57BL/6 and BALB/c mice were purchased from Charles River Laboratories
(Wilmington, MA). B6.PL-Thy1a/CyJ (Thy1.1 congenic), B6.PL-Thy1a/CyJ,
B6.129S2-Igh-6tm1Cgn/J (referred to as B2/2 mice), and B6/JiKbtm1Dbtm1N12
(referred to as KbDb2/2 mice) were obtained from The Jackson Laboratory
(Bar Harbor, ME). CD11c–diphtheria toxin receptor (DTR) mice were bred in
the Central Animal Facility at McMaster University. All animal experimentation was approved by McMaster University’s Animal Research Ethics Board
and complied with the Canadian Council on Animal Care guidelines.
ROLE OF SPLENIC APCs IN SECONDARY T CELL RESPONSES
The Journal of Immunology
FIGURE 1. VSV can rapidly induce robust
CD8+ T cells responses in a route- and dosedependent manner. (A) C57BL/6 mice were
primed with Ad-SIIN (1 3 108 PFU, i.m.).
Fourteen days later, they were treated with PBS
or boosted with VSV-SIIN (1 3 109 PFU) by
various routes. Five days after treatment with
VSV, OVA-specific CD8+ T cells in blood were
quantified by tetramer staining. (B) C57BL/6
mice were primed i.m. with Ad-hDCT and
boosted 14 d later with VSV-hDCT via different routes. DCT-specific CD8+ T cells in blood
were measured 5 d after treatment with VSV.
(C) C57BL/6 mice primed with Ad-hDCT
were boosted with VSV-hDCT or Maraba-hDCT
after a 14-d interval. Five days after boosting,
CD8+ T cell responses to DCT180–188 were
quantified by detection of intracellular cytokine
staining. All graphs show means with SE bars;
n = 5 per group; one-way ANOVA. Each
experiment was replicated a minimum of three
times. **p , 0.01, ***p , 0.001, ****p ,
0.0001.
that rhabdoviruses share similar biological properties (Fig. 1C).
Intravenous administration of VSV-hDCT also induced a dramatic
expansion of CD8+ T cells in BALB/c mice, indicating that VSVmediated boosting was not strain specific (Fig. 2A). We also determined that the VSV boost was effective in the same time frame
(i.e., 7–14 d after primary immunization) in mice primed with DCs
or VV expressing the same Ag (Fig. 2B, 2C). Thus, the remarkable
potency of the VSV boost was not limited to a single strain, Ag, or
priming method but did require i.v. delivery of the boosting virus.
The therapeutic relevance of this prime-boost approach was demonstrated by an aggressive murine melanoma model (B16) where a
single vector alone only slightly slowed down the growth of preestablished B16 tumors (5 d old) and mice reached the endpoint
rapidly in 12 d after treatment (Fig. 2D). However, priming animals
bearing 5-d-old tumors with either Ad or DCs was sufficient to allow
a boost by VSV 9 d after priming, leading to complete tumor regression (Fig. 2D).
Intravenous boosting with VSV promotes secondary expansion
of TCM within the spleen
To learn more about the mechanism of the VSV-mediated boost, we
first determined the locations of secondary expansion of T cells
following i.v. inoculation of VSV. Mice were primed with AdhDCT and boosted 14 d later by i.v. VSV-hDCT. As a control, a
subset of the primed mice received only VSV-MT, which does not
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(16, 18). Our observation that i.v. injection of rhabdoviruses also
resulted in a rapid and robust secondary expansion of Ag-specific
CD8+ T cells prompted us to determine whether i.v. delivery was a
particularly efficient route for booster vaccinations. Mice were
immunized with an Ad vector expressing an OVA-derived CD8+
T cell epitope (OVA257– 264, SIINFEKL) coupled to luciferase (AdSIIN) (26). Fourteen days following immunization, OVA-specific
CD8+ T cells had expanded to 12% of total circulating CD8+ T cells
(Fig. 1A, lower panel) and primarily displayed a TEFF phenotype
(CD62LloCD127loKLRG1higranzyme Bhi; data not shown). Despite
the high frequencies of TEFF, i.v. administration of a VSV vector
encoding the same Ag (VSV-SIIN) provoked a massive expansion
of OVA-specific cells, reaching a mean of 88% of circulating CD8+
T cells (Fig. 1A, lower panel) or a 58-fold increase by absolute
numbers (Fig. 1A, upper panel). However, other routes, including
i.m., i.p., intranasal, and s.c., were not as efficient as i.v. (Fig. 1A,
upper panel).
We next examined the route dependence of the boosting immunization using another Ag. In this case, mice were primed with an Ad
expressing the weakly immunogenic melanocyte-differentiation Ag
DCT (Ad-hDCT) and boosted with a VSV expressing the same Ag
(VSV-hDCT) (22). Interestingly, not only was i.v. injection the best
route for the booster vaccination, it was the only route that generated a significant boost against this self-epitope (Fig. 1B). Similar
boosting responses were achieved with Maraba virus, suggesting
4589
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ROLE OF SPLENIC APCs IN SECONDARY T CELL RESPONSES
express any transgene. The frequency of DCT-specific CD8+ T cells
was assessed in various lymphoid tissues on days 1, 2, 3, 4, and 7
following administration of VSV. To monitor local proliferation,
BrdU was administered to the mice for 24 h prior to lymphocyte
harvest. As shown in Fig. 3A, the proliferative response peaked
around days 3–4 following boosting and returned to baseline by
day 7. Notably, the earliest evidence of a proliferative response
was observed in the spleen at day 2 after boosting, suggesting that
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FIGURE 2. Efficient boosting is not limited to
a single mouse strain, Ag, or priming method.
(A) BALB/c mice were primed with Ad-hDCT
and then treated i.v. with PBS or boosted with
VSV-hDCT at a 14-d interval. Five days after
boosting, DCT-specific CD8+ T cells were
measured in blood by flow cytometry after
in vitro restimulation with pooled immunoreactive peptides and intracellular cytokine staining.
****p , 0.0001. (B) C57BL/6 mice that received DCs pulsed with the gp33 peptide were
boosted 14 d later with VSV-gp33. (C) C57BL/6
mice primed with VV-SIINFEKL-Luc were
boosted with VSV-SIINFEKL-Luc. Five days
after treatment with VSV, SIINFEKL-specific
CD8+ T cells were quantified by intracellular
cytokine staining. (D) C57BL/6 mice carrying
5-d-old B16-gp33 tumors were treated with DC/
gp33 or Ad-gp33 vaccine alone or in combination with VSV-gp33 at a 9-d interval. PBS or
VSV-gp33 alone was included as controls. Means
plus SE bars are shown; n = 5 per group; two-way
ANOVA. Most of these groups were repeated
more than once, with a similar result.
the secondary expansion began in the spleen somewhere between
24 and 48 h after boost. Ultimately, proliferation was observed in
all tissues, likely as a consequence of migratory activated CD8+
T cells. To further confirm that the spleen was the primary site
where the proliferation of secondary T cells started, we carried out
two more experiments. First, we treated mice with FTY720 to
inhibit lymphocyte egress from lymphoid organs during boosting,
and T cell proliferation was monitored by BrdU incorporation.
The Journal of Immunology
4591
Compared to the VSV-MT control, the earliest proliferation of
hDCT-specific CD8+ T cells was evident only in the spleen 48 h
after boosting with VSV-hDCT (Fig. 3A, 3B, Supplemental Fig.
1). Second, spleens in Ad-hDCT–primed mice were surgically
removed 2 d before VSV-hDCT boosting (i.e., 12 d after priming)
and secondary expansion was determined on day 5 after boosting.
Data in Fig. 3C show that CD8+ T cell expansion was abrogated in
the absence of a spleen, reinforcing a critical role of this organ in
initiating the secondary T cell responses.
The population of Ad-induced CD8+ T cells within the spleen on
the day of the boost was composed primarily of TEFF, although a
small population of TCM was also detectable (Fig. 4A). To determine
which CD8+ T cell subset was contributing to the secondary expansion, OVA257–264-specific splenic CD8+ T cells (Thy1.1) were
sorted into TEFF, TEM, and TCM subsets based on CD127 and CD62L
expression (Fig. 4B) (27). These subsets were adoptively transferred
into naive congenic recipients (Thy1.2) that were subsequently immunized with VSV-SIIN. Owing to the low frequencies of Agspecific CD8+ T cells in the TCM population, we did not attempt
to normalize the number of Ag-specific T cells that were transferred.
Rather, we normalized the total number of adoptively transferred
cells using naive splenocytes to ensure that equal numbers of total
T cells were delivered to each recipient (106 cells per mouse).
Tetramer staining indicated that each bolus of 106 cells contained
1.9 3 105 OVA257–264-specific TEFF, 6.6 3 104 TEM, or 4 3 103 TCM.
Frequencies of adoptively transferred cells (Thy1.1) were determined 5 d after boosting (Fig. 4C). The transgene-specific TCM
were the primary responders, as they underwent dramatic expansion
5 d after exposure to VSV-SIIN (i.e., 135-, 425-, and 206-fold increases in frequency relative to day 21 in the spleen, bone marrow,
and blood, respectively). In contrast, TEM contributed little to the
expansion (∼50% increase over input). As expected, TEFF did not
contribute to the secondary expansion and actually contracted
during the 5-d period. Our observation is consistent with previous
reports that the TCM possess the highest proliferative potential. The
results also demonstrated that the TCM subset was preferentially and
effectively expanded by the VSV vaccine.
Intravenous delivery of VSV results in B cell infection in the
spleen
Previous reports have indicated that Ag presentation is rapidly shut
off by CD8+ TEFF through a feedback mechanism that involves
killing Ag-loaded APCs (14, 28). To determine which cell types
were infected by VSV, a vector expressing GFP (VSV-GFP) was
injected i.v. into mice and spleens were harvested 90 min later to
minimize the migration of infected cells between tissues. Singlecell suspensions were incubated for an additional 4.5 h to provide
sufficient time for the VSV to express GFP and then infected cells
were analyzed by flow cytometry (Fig. 5A). Strikingly, most infected cells within the spleen were B220+CD19+ B cells (∼82%;
Fig. 5B). Further characterization of the VSV-infected B cells
revealed most were CD21/35intCD23hiCD272, which is indicative of follicular B cells (Fig. 5B) (29). Ag expression by B cells
within the follicular region may represent a unique mechanism to
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FIGURE 3. The spleen is the primary site for
secondary expansion of central memory T cells.
(A) Ad-hDCT–primed C57BL/6 mice were
boosted 14 d later with VSV-hDCT. Each group
received BrdU via i.p. injections and drinking
water 24 h prior to tissue harvest, at which time
BrdU+ DCT-specific CD8+ T cells were enumerated (n = 3 per group per time point; mean
with SE bars; two-way ANOVA; *p , 0.05). (B)
C57BL/6 mice primed with Ad-hDCT were
boosted with VSV-hDCT 14 d later. Twenty-four
hours after treatment with VSV, mice received
BrdU and FTY720 via i.p. injections. At 24 h
after BrdU/FTY720 treatment, harvested tissues (spleen, lymph nodes, and bone marrow)
were assessed by tetramer staining to quantify
DCT180–188-specific CD8+ T cells. (C) Mice
primed with Ad-hDCT received splenectomies
(Splx) or sham surgeries 12 d later. At 48 h after
surgery, mice were boosted with VSV-hDCT.
Five days after treatment with VSV, DCT180–188specific CD8+ T cell responses were quantified
by intracellular cytokine staining. Means plus
SE bars are shown; n = 5 per group; unpaired
t tests (B) and two-way ANOVA (C). This experiment was replicated three times.
4592
ROLE OF SPLENIC APCs IN SECONDARY T CELL RESPONSES
avoid TEFF-mediated clearance because TEFF cannot circulate
through this area (30). The GFP expression pattern in the spleen,
together with the fact that TCM were primarily reactivated in the
spleen, appeared to suggest that follicular B cells might be serving
as APCs for the secondary expansion.
B cells are required for optimal boosting by VSV, but direct Ag
presentation is mediated by DCs
To characterize the requirement of B cells in vivo for the secondary
expansion of TCM, we tested our prime-boost regimen in B cell–
deficient (B2/2) mice (31). WT and B2/2 mice were immunized
with Ad-hDCT and boosted 14 d later with VSV-hDCT. The absence of B cells had no effect on the primary response to the
DCT transgene carried by the Ad vector (Fig. 6A, 1˚). In contrast,
the secondary response to hDCT was severely attenuated in the
absence of B cells (Fig. 6A, 2˚), suggesting that B cells were
required for secondary CD8+ T cell expansion. Because B cells
are necessary for the organogenesis of lymphoid tissues (32), we
repeated the same experiment in WT mice where B cells were
depleted with an anti-CD20 mAb. Similar to the results in B2/2
FIGURE 5. Intravenous delivery of VSV results in B cell infection in the spleen. (A)
Spleens were harvested from C57BL/6 mice
that received i.v. injection of PBS (not shown)
or 1 3 109 PFU live or UV-inactivated VSVGFP for 1.5 h. Splenocytes were cultured for an
additional 4.5 h so that enough GFP could be
produced by infected cells to allow detection by
flow cytometry. (B) Infected (GFP+) cells were
stained for B220 and CD19 or CD11b and
CD11c to detect B and myeloid cells, respectively. VSV-GFP–infected splenic B cells were
further characterized based on their expression
of CD21/35 and CD23. This experiment was
replicated five times.
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FIGURE 4. TCM are preferentially and effectively expanded by the VSV vaccine. (A) A
typical CD127 versus CD62L staining profile
of Kb-OVA257–264 (Tet)–specific CD8+ T cells
from the blood of a C57BL/6 mouse 14 d after
Ad-SIINFEKL-Luc vaccination. (B) Splenocytes from congenic Ad-SIINFEKL-Luc–immunized mice (Thy1.1) were sorted 14 d later
into TEFF (CD1272CD62L2), TEM (CD127+
CD62L2), and TCM (CD127+CD62L+) T cell
subsets. (C) Pooled splenocytes from Ad-SIIN–
immunized congenic mice (Thy1.1) were used
to sort OVA-specific T cells into TEFF, TEM, and
TCM subsets. Total numbers of cells were normalized using naive splenocytes and then these
subsets were injected i.v. into naive C57BL/6
(Thy1.2) recipient mice. Twenty-four hours
later, mice received 1 3 109 PFU VSV-SIIN i.v.
Five days after treatment with VSV the frequencies of OVA-specific Thy1.1 T cells were
determined in various tissues and expressed as a
fold change over the frequencies present in the
cells that were originally transferred (n = 5 per
group; mean with SE bars; one-way ANOVA).
These experiments were replicated twice.
The Journal of Immunology
mice, the ablation of B cells before boosting with VSV had a
dramatic negative effect on the expansion of the memory DCTspecific T cells (Fig. 6B).
Having established a critical role for B cells in secondary expansion of TCM, we next investigated whether B cells were required for Ag presentation. We generated chimeric mice using
bone marrow cells from B2/2 and KbDb2/2 mice to reconstitute
lethally irradiated WT mice. In these chimeras, all B cells could
only be derived from the KbDb2/2 bone marrow whereas a large
proportion of other APCs (from the B2/2 bone marrow) had intact
MHC class I molecules (Supplemental Fig. 2). Similarly prepared
recipient mice reconstituted with WT and/or B2/2 bone marrow
were included as controls (Fig. 6C). Surprisingly, in contrast to the
results in B2/2 and B cell–depleted mice, the secondary expansion
was not attenuated when the B cells were unable to present Ag on
MHC class I (Fig. 6C), suggesting that B cells do not have a direct
role as an APC.
The above observations prompted us to determine whether
splenic DCs were responsible for Ag presentation. To address this
4593
possibility, we generated chimeric mice with bone marrow from
CD11c-DTR transgenic mice as described by Zammit et al. (12).
These mice tolerated multiple injections of DT to remove CD11c+
DCs (Supplemental Fig. 3). To ensure that CD8+ T cells were not
affected by DT treatment, we transferred Ag-primed CD8+ T cells
from WT congenic mice to CD11c-DTR chimeric mice prior to
boosting with VSV. Fig. 6D shows that injections of DT diminished expansion of endogenous T cells (Thy1.12) in CD11c-DTR
chimeric mice but not WT controls, suggesting that host DCs may
mediate Ag presentation to trigger memory T cell proliferation.
DT injections also reduced the secondary response of transferred
WT CD8+ T cells (Thy1.1+), confirming the requirement of
CD11c+ DCs instead of removal of certain CD8+ T cells that may
also express CD11c (Fig. 6E) (33, 34).
Taken together, these results pointed to a possibility that infected
B cells produced and transferred Ags to neighboring DCs, leading
to Ag presentation to TCM that were also localized in the follicular
region (20, 35). To confirm this possibility, we isolated B cells
from KbDb2/2 mice 17 h after i.v. inoculation with VSV-gp33 and
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 6. B cells are required for maximal CD8+ T cell expansion but DCs mediate direct Ag presentation. (A) C57BL/6 or B2/2 mice were immunized i.m. with 1 3 108 PFU Ad-hDCT and then boosted i.v. with 1 3 109 PFU VSV-hDCT 14 d later. DCT-specific CD8+ T cells were quantified in
blood at the peak of the primary (1o) and secondary (2o) immune responses (12 and 5 d after treatment with Ad and VSV, respectively). (B) C57BL/6 mice
with or without B cell depletion received Ad-hDCT followed by VSV-hDCT 14 d later. DCT-specific T cells were quantified in blood 5 d after treatment
with VSV. (C) Lethally irradiated C57BL/6 mice received bone marrow from WT and/or B2/2 and/or KbDb2/2 donors. Reconstituted mice were subsequently vaccinated i.m. with 1 3 108 PFU Ad-hDCT and boosted i.v. 14 d later with 1 3 109 PFU VSV-hDCT. DCT-specific CD8+ T cells were quantified
in blood at the peak of the primary (1o; 12 d after Ad) and secondary (2o; 5 d after VSV) immune responses. (D and E) Chimeras that had received bone
marrow grafts from WT (controls) or CD11c-DTR–transgenic mice were primed with 1 3 108 PFU Ad-hDCT i.m. and boosted 14 d later with 1 3 109 PFU
i.v. VSV-hDCT. Two days prior to injection of VSV, DCT-specific T cells isolated from spleens of congenic (i.e., Thy1.1+) Ad-hDCT–immunized mice
were adoptively transferred into the chimeras that had received CD11c-DTR bone marrow. One day prior to treatment with VSV and every other day
thereafter, half of the chimeric mice received 100 ng i.p. DT to deplete CD11c+ cells. Endogenous [Thy1.12 (D)] and adoptively transferred [Thy1.1+ (E)]
DCT-specific T cell responses in blood were quantified 6 d after VSV. In all cases, T cell responses were measured by detection of intracellular cytokines
following in vitro stimulation with peptides; n = 5 per group; means with SE bars; one-way ANOVA (B), two-way ANOVA (A and C), or unpaired t tests
(D and E). These experiments were replicated three times.
4594
coincubated them with CFSE-labeled P14 T cells in the presence
or absence of bone marrow–derived DCs. Three days after coculture, proliferation of P14 T cells was detected only in the
presence of DCs, suggesting that B cell–produced Ags were taken
by DCs and presented to T cells (Fig. 7A, upper panel). To confirm this, we used Transwell inserts to separate infected KbDb2/2
B cells (top of well) from P14 T cells and DCs (bottom of well).
P14 cell proliferation was observed only when DCs were present
in the lower compartment, confirming that Ag was transferred
from B cells to DCs in a soluble form (Fig. 7A, lower panel). To
determine whether infected B cells produce viral progeny that
reinfect DCs, we coincubated B cells from VSV-gp33–exposed
KbDb2/2 mice with Vero cells that are highly sensitive to VSV
replication. After 48 h, Vero cells coincubated with B cells
(Fig. 7B, middle panel) remained intact (compared with Vero
cells alone; Fig. 7B, left panel), whereas cytopathic effect was
visibly evident by direct infection with VSV (Fig. 7B, right
panel), suggesting that B cells neither permit productive viral
replication nor carry viruses on their surface.
TEFF-mediated elimination of Ag-laden DCs has been widely
accepted as a homeostatic mechanism to prevent overexuberant
activation of CD8+ T cells (5, 14, 28). We report in the present
FIGURE 7. Ags, in soluble form, can be transferred from infected
splenic B cells to DCs to induce T cell proliferation. (A) KbDb2/2 mice
were inoculated i.v. with VSV-gp33 for 17 h and splenic B cells were
purified. B cells were cultured with CFSE-labeled P14 cells in the presence
or absence of bone marrow–derived DCs, either directly (upper panels,
Transwell 2) or separated by a Transwell (lower panels, Transwell +).
Representative flow cytometry profiles of P14 cell proliferation at 72 h
after incubation are shown. (B) Five million purified B cells, prepared as
described above, were added to Vero cell monolayers and incubated for
48 h at 37˚C (middle). Vero cells alone (left) or infected with VSV (right)
were included as controls. These experiments were replicated twice.
Original magnification 350.
study, however, that i.v. delivery of rhabdoviral vectors could
overcome this negative regulation and maximize expansion of
CD8+ T cells, even at the peak of the primary response. A unique
cooperation between splenic B cells and DCs for Ag presentation
and their anatomic location (within the follicle), which is separated from TEFF but proximate to TCM, appears to be a mechanism
that enables engagement of TCM during an ongoing effector phase
in the presence of circulating viruses (i.e., viremia). Our data
revealed a distinct role of splenic APCs during secondary T cell
responses, which should be considered for the rational design of
booster vaccines to rapidly and effectively expand Ag-specific
CD8+ T cells. Our finding may also provide new fundamental
mechanistic insight into the immune response to viremia where
the primary CD8+ T cell response can be amplified to contend
with an uncontrolled virus infection without compromising the
regulatory feedback to DCs that limits further naive T cell recruitment and, presumably, minimizes inadvertent presentation of
tissue Ags by migrating DCs.
Previous research has demonstrated that the secondary lymphoid
organs are the primary sites of naive T cell priming following
immunization or pathogen infection. Upon activation, TEFF and
TEM enter the circulation and peripheral tissues, whereas TCM
home to the secondary lymphoid organs (36). This differential
distribution poses a potential barrier to interaction between Agladen DCs and TCM because cytolytic TEFF kill migratory DCs in
peripheral tissues (13, 15). Moreover, a recent study has shown
that TEFF can re-enter reactive lymph nodes and attenuate Ag
presentation by killing newly arrived DCs and Ag-loaded residential DCs (37). These data clearly demonstrate that the efficacy
of booster immunizations that rely on migratory DCs for Ag
presentation will be limited by this TEFF-mediated negative feedback mechanism, especially during primary and chronic immune
responses.
In the spleen, CD8+ TCM are located in the T cell zones of the
white pulp surrounded by follicular B cells. Using TCR-transgenic
CD8+ T cells, several groups have shown that after adoptive transfer to naive recipients, effector CD8+ T cells were only found in the
red pulp, whereas memory CD8+ T cells homed to the B cell follicles (19, 30, 38). Subsequent studies confirmed that this localization pattern held true for endogenously activated CD8+ T cells.
Lefrançois and colleagues (20) and Jacob and colleagues (21)
showed that upon secondary exposure to LCMV or Listeria monocytogenes, memory CD8+ T cells rapidly expanded and mobilized
from the B cell follicles to the red pulp via bridging channels. After
the immune response subsided, memory CD8+ T cells homed back
to the B cell follicles whereas effector cells remained in the splenic
red pulp (20, 21). These results point to a possibility that follicular APCs were responsible for memory CD8+ T cell activation.
Colocalization with TCM in the follicles confers upon follicular
APCs anatomical advantages to interact with TCM while avoiding
TEFF-mediated killing. In contrast, migratory DCs, including the
CD8a+ subset, primarily reside in the red pulp, making them highly
susceptible to killing by TEFF during secondary responses (39). Our
results provide unequivocal evidence that this negative regulation
can be bypassed when the Ag can be delivered into the follicular
region. This discovery has important implications for optimizing
therapeutic vaccination strategies where high numbers of TEFF are
needed for protection prior to boosting as well as in circumstances
where TEFF will inevitably be present for prolonged periods such as
during chronic infections.
It was previously demonstrated that delivery of VSV into the
flank leads to infection of the lymph nodes where most of the Ag is
taken up by macrophages and DCs within the subcapsular sinus, so
little of the virus gets into the B cell follicles (40). This may explain
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Discussion
ROLE OF SPLENIC APCs IN SECONDARY T CELL RESPONSES
The Journal of Immunology
Acknowledgments
We thank Drs. R. Dunn and M. Kehry at Biogen Idec (San Diego, CA) for
the gift of mAb against mouse CD20 (clone 18B12).
Disclosures
B.D.L. is a board member of Turnstone Biologics, which is developing
Maraba virus as an oncolytic vaccine. The other authors have no financial conflicts of interest.
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why immunization through discrete routes (e.g., s.c., i.m.), which
result in Ag uptake through the lymphatics, are not effective routes
for boosting. Additionally, the ability of VSV and Maraba virus
to infect splenic B cells, especially follicular B cells, not only bypasses circulating TEFF but also increases Ag production due to
their enormous number and subsequent Ag presentation by neighboring DCs to TCM, which are also located in the follicular region.
Our in vitro analysis confirmed that infected B cells could transfer
Ag to DCs without involving direct contact between the two cells
although the exact form of the Ag remains to be determined. It was
previously reported that B cells could carry viruses including VSV
on their surface and transfer them to other cells (41, 42), but our
results argue that the Ag is likely in a different soluble form.
Our data suggest a novel strategy for boosting immune responses
in the presence of TEFF by delivering Ags to a location that is anatomically separated from the TEFF and in close proximity to TCM.
These findings are supported by recent publications revealing differential distributions of TCM and TEFF and offer important insights
into how to rapidly boost immunity without the need to compromise the TEFF population induced by primary immunization.
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