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Memory CD8+ T Cell Responses Expand
When Antigen Presentation Overcomes T
Cell Self-Regulation
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of June 11, 2017.
Ian A. Cockburn, Sumana Chakravarty, Michael G.
Overstreet, Adolfo García-Sastre and Fidel Zavala
J Immunol 2008; 180:64-71; ;
doi: 10.4049/jimmunol.180.1.64
http://www.jimmunol.org/content/180/1/64
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References
The Journal of Immunology
Memory CD8ⴙ T Cell Responses Expand When Antigen
Presentation Overcomes T Cell Self-Regulation1
Ian A. Cockburn,* Sumana Chakravarty,* Michael G. Overstreet,* Adolfo Garcı́a-Sastre,†‡
and Fidel Zavala2*
T
he generation and expansion of protective T cell responses are the goals of vaccines being developed to fight
a variety of diseases including malaria, HIV, and tuberculosis (1–3). The usefulness of generating protective CD8⫹ T
cell responses has led to intense interest in understanding the
cellular requirements for effective recall and boosting responses. These studies have highlighted requirements for dendritic cells (DCs)3 and CD4⫹ T cells for CD8⫹ T cell expansion
(4 – 6) and have identified subsets of T cells that play important
roles in protection against a variety of pathogens (7–9). Nonetheless, one observation that has not, to our knowledge, been
thoroughly addressed in the recent literature is the finding that
memory T cell populations, once established, do not readily
expand further (10, 11). This is clearly a major obstacle to any
vaccine strategy aimed at inducing large numbers of protective
T cells. Many of the studies described above have overcome
this problem either by using secondary doses of Ag several
orders of magnitude larger than the primary immunization or by
using adoptive transfer protocols in which small numbers of
*Department of Molecular Microbiology and Immunology, Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205; and
†
Department of Microbiology and ‡Emerging Pathogens Institute, Mount Sinai
School of Medicine, New York, NY 10029
Received for publication May 25, 2007. Accepted for publication October 17, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AI044375 (to F.Z.)
and the National Institutes of Health-funded Center to Investigate Viral Immune Antagonism Grant U19AI62623 (to A.G.-S.).
memory cells, often of a single specificity, are transferred to an
otherwise naive mouse before boosting. Although valuable information has come from these studies, neither of these protocols is useful for expanding T cell responses by vaccination.
For the purposes of vaccination, prime-boost regimens (using
different vectors for the prime and the boost) have been identified
as the best way to expand CD8⫹ T cell responses (12). However,
unlike Ab responses, which are generally robustly boosted following re-exposure to Ag, few prime-boost combinations result in
strong CD8⫹ boosting (11, 13). This was illustrated in a study
using recombinant influenza and vaccinia vectors carrying the murine malaria epitope SYVPSAEQI. It was shown that robust protective CD8⫹ T cell responses could be generated after an influenza-prime/vaccinia-boost combination; however, when the
viruses were given in the reverse order a much smaller and nonprotective CD8⫹ T cell response was seen (14). We took advantage of this well-established malaria sporozoite model of CD8⫹ T
cell immunity to examine the cellular processes involved in expanding memory CD8⫹ T cell responses. By comparing the processes of Ag presentation after priming and boosting with various
vectors that all carry the protective SYVPSAEQI epitope, we
found that boosting occurred when Ag was targeted efficiently to
DCs. If insufficient Ag was made available to DCs, Ag presentation, and consequent T cell boosting, was rapidly limited by the
cognate CD8⫹ T cells themselves. Thus, according to these data,
T cell expansion can occur when the degree of Ag presentation is
sufficient to overcome self-regulation by CD8⫹ T cells.
Materials and Methods
Mice
2
Address correspondence and reprint requests to Dr. Fidel Zavala, Department of
Molecular Microbiology and Immunology, Malaria Research Institute, Johns Hopkins
Bloomberg School of Public Health, Baltimore, MD 21205. E-mail address:
[email protected]
3
Abbreviations used in this paper: DC, dendritic cell; Ad-CS, adenovirus-CS; FluME, influenza-ME; ␥-spz, irradiated sporozoites; VV-SYV, vaccinia-SYV; DT, diphtheria toxin.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
Female BALB/c mice, 5– 8 wk old, were purchased from Taconic. Transgenic mice expressing a TCR specific for the Plasmodium yoelii epitope
were derived as previously described (15). Mice from our colony that have
previously been backcrossed to the Thy1.1⫹ BALB/c background for ⬎20
generations were used. CD11c-DTR mice on a BALB/c background were
derived as described and are maintained in our colony as heterozygotes
(16). Experiments involving mice were approved by the institutional animal care and use committee of the Johns Hopkins University.
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Antimicrobial memory CD8ⴙ T cell responses are not readily expanded by either repeated infections or immunizations. This is
a major obstacle to the development of T cell vaccines. Prime-boost immunization with heterologous microbes sharing the same
CD8ⴙ epitope can induce a large expansion of the CD8ⴙ response; however, different vectors vary greatly in their ability to boost
for reasons that are poorly understood. To investigate how efficient memory T cell expansion can occur, we evaluated immune
regulatory events and Ag presentation after secondary immunization with strong and weak boosting vectors. We found that
dendritic cells were essential for T cell boosting and that Ag presentation by these cells was regulated by cognate memory CD8ⴙ
T cells. When weak boosting vectors were used for secondary immunization, pre-established CD8ⴙ T cells were able to effectively
curtail Ag presentation, resulting in limited CD8ⴙ T cell expansion. In contrast, a strong boosting vector, vaccinia virus, induced
highly efficient Ag presentation that overcame regulation by cognate T cells and induced large numbers of memory CD8ⴙ T cells
to expand. Thus, efficient targeting of Ag to dendritic cells in the face of cognate immunity is an important requirement for T cell
expansion. The Journal of Immunology, 2008, 180: 64 –71.
The Journal of Immunology
65
adoptively transferred into mice, it was possible to track these cells using
anti-Thy1.1 (clone His51; BD Biosciences). Proliferation of transgenic
cells in vivo or in vitro could be assessed by labeling with 0.6 ␮M CFSE
using the Vybrant Cell Tracker kit according to the manufacturer’s instructions (Invitrogen Life Technologies). ELISPOTs to measure SYVPSAEQIspecific IFN-␥-secreting cells were performed as described (19).
Cell isolation
FIGURE 1. CD8⫹ T cell memory is not substantially increased by repeated exposure to sporozoites: Mice were immunized at weekly intervals
with 1 ⫻ 105 ␥-spz according to different schedules given on the y-axes.
Ten days after the final immunization, the SYVPSAEQI-specific immune
response was measured either by tetramer staining (A) or by ELISPOT
analysis (B). Data are the mean and SE based on four mice per group.
Immunizations
Quantification of SYVSAEQI-specific T cell number and
proliferation
The Ag-specific response to the SYVPSAEQI epitope was measured by
tetramer staining and ELISPOT. PE-labeled SYVPSAEQI-specific H2-Kd
tetramers were prepared as previously described (18). Spleen cells were
incubated with tetramer and anti-CD8-APC (eBioscience) before FACS. In
experiments in which SYVPSAEQI-specific transgenic cells had been
Ag presentation assay
To assess Ag presentation ex vivo, splenic myeloid DCs were purified as
described in Cell isolation at various times postimmunization. DCs (5 ⫻
104) were mixed with an equal number of purified naive CFSE-labeled
CD8⫹-transgenic cells in a single V-bottom well of a 96-well plate. From
8 to 16 cultures were set up for each DC population; 60 – 65 h later, the
cells were harvested, and CFSE dilution in the transgenic cell population
was used as a measure of Ag presentation. The amount of Ag presentation
was expressed either as the percentage of cells that have divided (% M1 in
Fig. 4A) or as the percentage of cells that have entered division. This was
calculated by determining the number of cells in each generation peak and
dividing by 2n (where n is the generation number) to determine how many
naive cells contributed to that peak. The percentage of the original naive
population that entered division could then be calculated. From this was
subtracted the percentage of cells that entered division when incubated
with DCs from a naive animal. Values ⬍0.5% were considered below the
limit of detection.
FIGURE 2. VV-SYV boosts ␥-spz-immune responses. Mice were immunized with 1 ⫻ 105 ␥-spz and 21 days later were boosted with one of 1 ⫻ 107
Ad-CS, 2.5 ⫻ 103 Flu-ME, or 2 ⫻ 106 VV-SYV (all given i.v.) or left unboosted. Naive mice were also immunized with the three viral vectors alone. The
SYVPSAEQI-specific immune response was measured by tetramer staining at day 10 (A) or 21 (B) after the final immunization. Data are the mean and
SE based on three mice per group. C, The boosting factors (defined in text) of the various viral vectors derived from day 10 (u) and day 21 (f) data in
A and B. D, Mice were immunized with 1 ⫻ 105 ␥-spz and 21 days later were boosted with 6.25 ⫻ 104 Flu-ME or left unboosted. Naive mice were also
immunized with Flu-ME alone. The SYVPSAEQI-specific immune response was measured by tetramer staining at day 10. Data are the mean and SE based
on three mice per group.
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Plasmodium yoelii strain 17X NL sporozoites were obtained and irradiated
as previously described (15). The generation of the vaccinia-SYV (VVSYV), influenza-ME (Flu-ME), and adenovirus-CS (Ad-CS) have been
described previously (13, 14, 17): the influenza virus carried the SYVPSAEQI epitope engineered into viral hemagglutinin; VV-SYV carries the
epitope integrated into the viral thymidine kinase gene and Ad-CS has
the entire CS gene under the control of the E1 gene promoter, meaning
that the it will be essentially the only adenovirus-derived gene product
produced in infected cells. Doses used were 1 ⫻ 104 ␥-spz, 2 ⫻ 106
VV-SYV, 2.5 ⫻ 103 Flu-ME, and 1 ⫻ 107 Ad-CS; all immunizations were
given i.v. Ad-CS was kindly provided by Moriya Tsuji (New York University, New York, NY).
Single-cell suspensions of lymphocytes were obtained by grinding spleen
cells between the ground ends of two microscope slides and filtering twice
through 100-␮m pore size nylon mesh. Where necessary, untouched purified populations of CD8⫹ T cells were obtained using the mouse CD8⫹ T
cell isolation kit according to the manufacturer’s instructions (Miltenyi
Biotech). Splenic myeloid DCs were prepared essentially as described (20).
Briefly, spleens from mice immunized or naive mice were taken, chopped
finely and digested with 1 mg/ml collagenase. The liberated cells were spun
through FCS supplemented with EDTA. The spleen cells were separated
over a Nycodenz gradient (density, 1.075 g/ml) and the DC-rich low-density fraction was taken. Contaminating non-DC populations were stained
with FITC-conjugated anti-CD3, anti-Thy1.2, anti-GR1, anti-B220, and
anti-TER119 Abs (all from eBioscience) and depleted by magnetic bead
separation using anti-FITC microbeads according to the manufacturer’s
instructions (Miltenyi Biotec). This yielded a 60 – 80% pure MHC class
II⫹CD11c⫹ DC population as determined by FACS.
66
MECHANISM FOR CD8⫹ T CELL BOOSTING
Cell depletion
DCs were depleted from CD11c-DTR-transgenic mice by 2 i.p. injections
of 100 ng of diphtheria toxin 3 days apart. This resulted in ⬃90% depletion
of DCs for ⬃120 h. BALB/c mice are able to accept this dose without
weight loss or the mortality seen in C57BL/6 mice (6). CD8⫹ cells were
depleted by 2 i.p. injections on consecutive days with 200 ␮g of anti-CD8
Ab (clone 2.43), whereas CD4⫹ cells were depleted by two i.p. injections
on consecutive days of 200 ␮g of anti-CD4 Ab (clone GK1.5).
Data acquisition and analysis
FACS data were acquired on a FACSCalibur machine and analyzed using
FlowJo software (TreeStar). ELISPOT plates were read with an Immunospot plate reader, and the data were analyzed using Immunospot software
(Cellular Technology). Graphs were prepared, and statistical analysis was
performed using GraphPad Prism (GraphPad Software).
Results
CD8 T cell expansion is not an automatic consequence of
re-exposure to Ag
A vaccinia vector is capable of expanding memory CD8 T cells
One explanation for the previous result is that immunity develops
against ␥-spz which will clear the parasite on re-exposure and thus
limit its ability to further expand immune responses. For this reason, heterologous prime-boost vaccinations are used to expand
CD8⫹ T cells. We therefore tried to boost a single dose of sporozoites with recombinant Ad-CS, VV-SYV, or Flu-ME, all of which
carry the protective SYVPSAEQI epitope (13, 14, 23). These vectors have all been extensively described previously; in this system,
the vectors were given i.v. for consistency, although all have been
shown to elicit protective CD8 T cells regardless of the route of
immunization (23, 24). The epitope-specific immune responses to
the different prime-boost combinations and the component immunizations alone were measured with H2-Kd tetramer at days 10 and
21 (Fig. 2, A and B). To normalize results between the different
vectors, we chose doses of virus that gave approximately equivalent priming responses (Fig. 2, A and B) and defined a simple
boosting factor as (prime;boost)/(prime alone ⫹ boost alone).
Surprisingly, given that they all induce similar priming responses, Ad-CS and Flu-ME were capable of boosting Ag-specific
responses only to ⬃1–2 ⫻ 105 tetramer⫹ cells/spleen (Fig. 2, A
and B) which corresponded to a modest boosting factor of ⬃2 (Fig.
2C). In experiments in which a 25-fold higher dose of Flu-ME
(6.25 ⫻ 104) was used to boost sporozoite-immunized mice, we
observed only a modest increase in the boosting factor to 2.7 (Fig.
2D). VV-SYV immunization, however, resulted in strong boosting
with tetramer⫹ cells expanding to ⬎5 ⫻ 105 cells/spleen, corresponding to a boost factor between 5 and 7 (Fig. 2, A–C). Similar
data were obtained when we measured specific responses by ELIS
POT (data not shown). We therefore found that in this system not
all heterologous prime-boost combinations result in significant
FIGURE 3. Cellular requirements for boosting. A, DCs are required for
boosting. 1 ⫻ 105 memory Thy1.1-transgenic cells (purified from the
spleens of mice that had received transgenic cells and been primed with
2 ⫻ 105 ␥-spz) were adoptively transferred into CD11c-DTR transgenic
mice on a BALB/c, Thy1.2 background (i), or wild-type mice (ii). The
mice were then left unboosted; boosted with VV-SYV 1 day later, or DC
depleted and boosted with VV-SYV. Five days after boosting, the spleen
cells were harvested, and transgenic cells were identified by staining for
Thy1.1 and CD8. Plots show representative FACS data from each group.
The values are the mean and SD of the percent of CD8⫹ Thy1.1⫹ cells
based on three mice per group. B, CD4⫹ T cells are not required for boosting. Mice were immunized with ␥-spz and boosted 21 days later with
VV-SYV with or without depletion of CD4⫹ T cells 2 days before boosting. Other mice received the individual immunizations alone. The SYVP
SAEQI-specific immune response was measured either by tetramer staining (i) or ELISPOT (ii) 12 days (f) and 28 days (u) after boosting. Data
are means and SEs based on three mice per group; differences between
anti-CD4 treated and control mice were not significant in any instance.
CD8⫹ T cell expansion and that vaccinia vectors have the unusual
property of boosting specific CD8⫹ T cell responses. These observations are consistent with our own previous data and data from
various other laboratories working on a variety of systems (3,
25–28).
Boosting by VV-SYV requires DCs but not CD4⫹ T cells
Given that vaccinia was most capable of inducing memory CD8⫹
T cell expansion, we investigated the cellular requirements for the
T cell expansion induced by this vector. To determine whether
VV-SYV boosting required DCs, we took advantage of the
CD11c/DTR transgenic mouse system in which the diphtheria
toxin (DT) receptor is expressed under the control of the CD11c
promoter; therefore, DCs may be depleted by injection with small
amounts of DT (16). SYVPSAEQI-specific memory CD8⫹ T
cells, carrying the Thy1.1 allele, were transferred into naive DTR
(Thy1.2)-transgenic mice. These mice were boosted with VV-SYV
with or without concurrent DC depletion. To deplete DCs, DT
(100 ng/mouse) was administered twice: 1 day before boosting;
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Despite continuous exposure to infectious bites, individuals living
in endemic areas develop CD8⫹ T cell responses of low magnitude
to sporozoite Ags (21, 22). This may be because CD8⫹ T cell
responses are hard to expand once they have been established (10).
We modeled natural exposure to malaria Ag by giving weekly
doses of P. yoelii 17X NL-␥-spz and measuring the Ag-specific
CD8⫹ T cell response by tetramer staining or ELISPOT (Fig. 1).
We found that giving four weekly doses of Ag gave an immune
response no larger than a single dose of Ag. Even leaving 3 wk
between two immunizations did not lead to a large expansion of
the CD8⫹ T cell population. Our data therefore suggest that CD8⫹
T cell responses are unlikely to significantly expand among individuals in an endemic area who may receive weekly or even daily
exposure to parasites.
The Journal of Immunology
and 2 days after boosting; Six days after boosting, the expansion of
memory Thy1.1 T cells was measured by FACS. In the DC-depleted mice, the cells expanded to one-sixth of the level seen in
intact mice (Fig. 3Ai). Importantly, DT treatment had no effect on
memory T cell expansion in normal mice, showing that the observed effect in CD11c/DTR mice was not due to any nonspecific
toxicity (Fig. 3Aii). Thus, DCs are required for efficient boosting of
immune responses by VV-SYV. In contrast, depletion of CD4
cells immediately before boosting did not result in a significant
reduction in the size of the boost as measured by either ELISPOT
or tetramer staining, either 12 or 28 days after boosting (Fig. 3B).
Ag presentation is slower and less efficient in Flu-ME- and
Ad-CS-immunized mice compared with VV-SYV-infected mice
Because boosting by VV-SYV requires DCs, we decided to investigate further the process of Ag presentation by DCs after immunization with the different vectors. We wished to determine
whether there were differences in this respect between the boosting
vectors and nonboosting vectors. To measure Ag presentation,
splenic DCs were purified from immunized mice and incubated
with an equal number of CFSE-labeled transgenic cells; Ag pre-
FIGURE 5. VV-SYV is able to prime SYVPSAEQI-specific transgenic
cells in the presence of ␥-sporozoite immunity. Naive CFSE-labeled
SYVPSAEQI-specific transgenic cells (2–3 ⫻ 106) were adoptively
transferred into either naive mice or ␥-spz-immune mice. One day later,
the mice were immunized with either VV-SYV, Flu-ME, or AdCS. Ten days later, spleen cells were harvested and stained with antiThy1.1 and anti-CD8 to identify transgenic cells. A, Numbers of
Thy1.1⫹CD8⫹CFSElow cells (expressed as a percent of total CD8⫹ T cells)
in the spleen after immunization with each of the vectors in previously
naive mice (u) and preimmune mice (f); values are means and SEs of
three mice per group; ns, not significant; ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 by
Student’s t test. B, Representative CFSE profiles from each group of mice;
plots were gated on Thy1.1⫹, CD8⫹ cells. Values are the mean and SE of
the percent of transgenic cells that have fully retained the CFSE label (M2)
based on three mice per group; p values are the differences between the
percent of cells retaining the CFSE label in naive and ␥-spz-immune mice
by Student’s t test. Data are from one of two experiments that yielded
similar results.
sentation was inferred from the dilution of the label after 60 – 65 h.
Transgenic cells incubated with spleen DCs from naive control
mice underwent a small amount of background proliferation (Fig.
4A). VV-SYV induced very efficient Ag presentation with ⬎80%
of transgenic cells dividing within 24 h of immunization (Fig. 4B).
Using the CFSE peaks to infer the number of divisions that each
cell has undergone, we estimate that ⬃30% of the transgenic cells
entered division. This pattern persisted for at least another 48 h
before beginning to wane by 120 h. Ag presentation by DCs after
Flu-ME immunization followed a very different course, despite the
fact that immunization with this vector results in a immune response of a size similar to that induced by VV-SYV (Fig. 4B). Ag
presentation was not detectable at 24 h and only began to be seen
at 48 h (data not shown) before peaking at 72 h postimmunization.
However, the peak response was much more limited with only
10 –20% of T cells dividing corresponding to only ⬃1–3% of the
original population having entered division. Similarly, immunization with Ad-CS resulted in Ag presentation which probably
peaked at ⬃72–120 h after immunization, although the overall
amount was near the limit of detection (Fig. 4B).
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FIGURE 4. Ag presentation after immunization by Flu-ME, Ad-CS,
and VV-SYV. A, CFSE profile of transgenic cells after incubation with
DCs from naive mice, value at top left is the percent of cells that have
divided (M1). B, Time course of Ag presentation after immunization of
naive mice with our standard doses of VV-SYV, Flu-ME, and Ad-CS. Data
are based on pooled DCs from three mice per group; values at top left are
the percent of cells that have divided (M1) and (numbers in parentheses)
the percent of cells entering division. Panels show representative data of at
least two independent experiments per vector. C, Priming by Flu-ME but
not VV-SYV is limited by CpG treatment 24 h before immunization. Mice
were treated with 10 nmol CpG/mouse 24 h before immunization to block
Ag cross-presentation. Ten days after immunization, the Ag-specific immune response was determined by tetramer. Data are means and SEs of
three mice per group; ns, not significant; ⴱⴱ, p ⬍ 0.01 by Student’s t test.
Representative data from one of two independent experiments are shown.
67
68
MECHANISM FOR CD8⫹ T CELL BOOSTING
We were interested in how VV-SYV could induce such a different pattern of Ag presentation compared with Flu-ME and AdCS. Vaccinia virus is known to infect DCs and induce direct presentation of Ag (29 –31). In contrast, as indicated by recent studies,
normal immune responses to influenza virus appear to develop
when viral Ag is available to DCs by cross-presentation (32). The
hypothesis that direct infection and Ag presentation plays a role in
the induction of CD8⫹ T cell responses by VV-SYV was tested by
blocking cross-presentation by systemic administration of a TLR
ligand (20). CpG treatment 24 h before immunization did not
dampen VV-SYV-induced immune responses. In contrast, priming
by Flu-ME was strongly abrogated by prior CpG treatment (Fig.
4C). It is possible that CpG may be inducing antiviral mechanisms
that affect Flu-ME Ag production; however, recent studies using
HSV have showed that CpG treatment blocks viral Ag cross-presentation without significantly reducing viral load (20). These data,
along with the previously published work described above (20,
29 –32), suggest that the different patterns of Ag presentation by
DCs after Flu-ME and VV-SYV immunization may result from
vaccinia infecting DCs and targeting Ag for efficient presentation
early in infection.
VV-SYV is able to target Ag for presentation and prime
transgenic cells in the presence of pre-existing CD8⫹
sporozoite immunity
Robust early Ag presentation by DCs after VV-SYV immunization
could contribute to its property of boosting CD8⫹ T cell responses.
It has been previously shown that Ag presentation of a given Ag is
rapidly limited by feedback regulation by the recently primed
CD8⫹ T cells (33–35). The effector cells either outcompete naive
cells for presented Ag, or simply clear Ag before all naive cells
have a chance to be activated; thus, there is a natural limitation on
the size of CD8⫹ T cell responses. A similar process is likely to
occur during secondary immunization to limit T cell expansion.
Boosting, however, could occur if there is efficient presentation of
Ag, and many memory cells have a chance to see their cognate
epitope on DCs before Ag presentation wanes. Because VV-SYV
makes large amounts of Ag available on DCs, we hypothesized
that it, unlike the other vectors, may be able to overcome feedback
regulation. To test this, we compared the ability of the different
vectors to drive the expansion of naive CFSE-labeled transgenic
cells in ␥-spz-immunized mice. In agreement with the hypothesis,
VV-SYV was able to prime transgenic cells in both naive and
␥-spz-immunized mice: in both cases a significant increase in the
number of cells was seen (Fig. 5A), and the CFSE label was diluted
out completely (Fig. 5B). In contrast, we saw evidence of feedback
regulation when we immunized ␥-spz-immune mice with Flu-ME
and Ad-CS; in both cases, there was significantly less expansion of
the transgenic cell population than with immunization in naive
control mice (Fig. 5A), and many cells retained the CFSE label
(Fig. 5B).
To further investigate the relative ability of VV-SYV and
Flu-ME to overcome regulation in immune mice, we compared
Ag presentation by DCs after Flu-ME and VV-SYV immunization in naive and ␥-spz-immune mice. Because of the limited
Ag presentation seen after Ad-CS immunization, it was not possible to include this vector in these experiments. Twenty-four
hours after booster immunization by VV-SYV, we saw no reduction in Ag presentation compared with that seen after a priming
immunization by this vector (Fig. 6A). Seventy-two hours after
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FIGURE 6. Ag presentation in sporozoite-immune mice. Preimmune and naive mice were immunized with either VV-SYV (A) or Flu-ME (B), and Ag
presentation was measured 24 and 72 h later. In some groups of mice, CD8⫹ cells or CD4⫹ cells were depleted 14 days before boosting. Data in all panels
are based on pooled DCs from three mice per group; values in each panel are the percentage of the total transgenic pool that have undergone at least one
division (M1) and (numbers in parentheses) the estimated percentage of the original naive population that entered division. Data are representative of four
similar independent experiments.
The Journal of Immunology
FIGURE 7. Ag presentation in mice after immunization with a high
dose of Flu-ME. Naive and preimmune mice were immunized with 6.25 ⫻
104 Flu-ME, and Ag presentation was measured 24 and 72 h afterward.
Data are based on pooled DCs from three mice per group; values are the
percent of the total transgenic pool that has undergone at least one division
(M1) and (numbers in parentheses) the estimated percentage of the original
naive population that entered division.
Boosting responses are regulated by CD8⫹ T cells
We have shown that Ag presentation by Flu-ME is severely curtailed in ␥-spz-immunized mice. There was also a small reduction
in Ag presentation after VV-SYV immunization in immune mice
compared with naive mice. Given that the only known shared immunological determinant between ␥-spz and these viral vectors is
the SYVPSAEQI epitope, we have hypothesized that feedback
regulation by CD8⫹ T cells mediated these effects. To test this
formally, we tried to recover Ag presentation in immune mice to
levels seen in naive mice by removing their CD8⫹ T cells by Ab
depletion. Two weeks after Ab depletion, the mice were boosted
with Flu-ME or VV-SYV, and Ag presentation was measured.
With both viral vectors, Ag presentation was recovered in sporozoite-immune mice to a level comparable with that seen in naive
mice when the mice were depleted of CD8⫹ T cells (Fig. 6). In
contrast, immune mice with depleted CD4⫹ T cells still showed
reduced Ag presentation as in untreated ␥-spz-immune mice
(Fig. 6).
Finally, the hypothesis that CD8⫹ T cells limit boosting, either
by clearing viral infection from the periphery or by limiting the
capacity of DCs to present Ag, was further tested by transferring
different numbers of transgenic memory CD8⫹ T cells into normal
mice which were subsequently boosted with VV-SYV. After 10
days, the size of the boost determined and the fold change in the
number of cells were measured. In agreement with the hypotheses,
the more cells transferred, the smaller was the fold change in the
number of cells (F ⫽ 24.1; p ⬍ 0.01 by one-way ANOVA; Fig. 8).
FIGURE 8. Memory CD8⫹ T cells regulate their own boosting. Different numbers (2 ⫻ 104; 1 ⫻ 105; 5 ⫻ 105) of memory CD8⫹Thy1.1⫹
SYVPSAEQI-specific transgenic cells were transferred into naive mice and
boosted with VV-SYV. Ten days later, the spleens were harvested, and the
size of the expanded transgenic population was measured by FACS. A, The
absolute number of Thy1.1⫹CD8⫹ cells recovered. B, The ratio of cells
recovered after boosting to cells transferred. All data are means and SEs
based on three mice per group.
Thus, the more memory cells present before secondary immunization, the greater is their ability to limit their own boosting on a
per cell basis. Overall, our data suggest that cognate CD8⫹ T cells
restrict boosting but efficient Ag presentation by DCs may overcome this to permit boosting.
Discussion
In this study, we wished to determine the cellular requirements for
T cell expansion and the characteristics required for a vector to
efficiently boost T cell responses. We believe this is the first systematic attempt to determine why some recall responses result in T
cell expansion and others do not. It was found that DCs are crucial
for the expansion of T cells after vaccinia boosting. CD4⫹ T cells
appear not to play a major role at the time of secondary immunization; rather, the most important regulators of CD8⫹ T cell expansion appear to be the cognate CD8⫹ T cells themselves. These
cells limit Ag presentation after secondary immunization with adenovirus and influenza virus vectors, resulting in limited CD8⫹ T
cell expansion. CD8⫹ T cell regulation, however, appears to be
overcome by the ability of VV-SYV to efficiently target Ag for
presentation by DCs. The resulting Ag presentation can induce
large numbers of memory CD8⫹ T cells to expand. The ability to
induce effective Ag presentation by DCs in the face of cognate
immunity could be one of the most important determinants of the
capacity of a vector to boost CD8⫹ T cell responses.
To our knowledge, this is the first description of cognate memory CD8⫹ T cells limiting their own recall responses. The role of
early effector CD8⫹ T cells in limiting priming of naive cells,
however, has been described previously by our group and others
(10, 33, 34). In these cases, Ag presentation appears to be limited
by the first wave of effector T cells, which may inhibit DC presentation of cognate Ag by a variety of means. Effector T cells may
directly kill DCs presenting cognate Ag (36 –38), strip peptide
from surface MHC molecules (39), down-modulate MHC class
I-peptide complexes on APCs (40), or outcompete naive cells for
Ag on the surface of DCs (41). Potentially, memory cells might
also use similar mechanisms to control boosting responses. Recently, it has been shown that effector and effector memory cells,
which are normally excluded from resting lymph nodes, can enter
reactive (inflamed) lymph nodes in a CXCR3-dependent manner
and kill DCs (42). Additionally, memory CD8⫹ T cells might clear
Ag in the periphery, preventing it from being cross-presented in
the lymphoid tissues. Together, these mechanisms allow effector
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booster immunization, there is still Ag presentation, although it has
declined compared with that seen in primed naive mice by between
20 and 80% in independent experiments (mean, 40%). The level of
variation was perhaps due to differences in the immunogenicity of
batches of sporozoites used for immunization in independent experiments. In Flu-ME-boosted mice, no Ag presentation was detected 24 h after boosting; whereas 72 h after secondary immunization with Flu-ME, Ag presentation is reduced by ⬃70%
compared with Ag presentation after priming (Fig. 6B). Ag presentation after boosting with a 25-fold higher dose of Flu-ME was
also strongly abrogated at 72 h (Fig. 7), although this dose did give
some early Ag presentation in immune mice, allowing it to stimulate a modest T cell expansion (Fig. 1D). Taken together, our in
vivo and ex vivo Ag presentation data strongly suggest that Ag
availability is a crucial limiting factor that may prevent some vectors from being able to boost immune responses.
69
MECHANISM FOR CD8⫹ T CELL BOOSTING
70
cinia to generate responses against HIV epitopes showed that the
attenuated virus was in fact the better boosting vector (26, 27).
Finally, it was surprising to us that Ad-CS did not boost more
efficiently in our system, since adenoviruses have previously been
suggested to be efficient boosting vectors (53, 54). However, it is
also known that adenovirus vectors induce very high primary responses. It may be that if the degree of boosting in previous studies
had been normalized to allow for this, as we have done with our
boosting factor, it would be found that the intrinsic ability of adenovirus to boost is not so pronounced. It is also compatible with
our hypothesis that giving several orders of magnitude more adenovirus or influenza virus might induce more efficient Ag presentation and therefore boosting. However, for vaccination, the immunizing dose is limited by the pathogenicity of the vector.
Nonetheless, it is important to emphasize that in our experiments
we did not set out to describe novel prime-boost combinations or
to suggest the use of one vector over another; rather, we aimed to
describe the basic cellular events involved in memory T cell
expansion.
There is an urgent need for new vaccines to fight the spread of
HIV, malaria, and tuberculosis as well as a host of so-called neglected diseases. To do this, we need a fundamental understanding
of how to induce and expand populations of protective memory T
cells. Our work shows that the ability of a vector to target Ag early
to DCs in the face of cognate immunity is vital for boosting T cell
responses. If these properties can be engineered into other vectors,
this could be a path to developing better vaccines designed to
induce protective CD8⫹ T cell responses.
Acknowledgment
We thank Nevil Singh (National Institute of Allergy and Infectious Diseases, Bethesda, MD) for his comments on the manuscript.
Disclosures
The authors have no financial conflict of interest.
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and memory CD8⫹ T cells to be powerful regulators of their own
priming and boosting.
It is clear from our experiments and those of others (6) that DCs
are required for the optimal expansion of CD8⫹ T cells during
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We have shown that VV-SYV makes ample Ag available on
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6A); however, why does this efficient Ag presentation not result in
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size of the vaccinia virus. Compared with Flu-ME, which produces
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CD8⫹ epitopes (44, 45). Robust presentation of the SYVPSAEQI
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antigenic complexity.
There may be other properties of vaccinia virus, influenza virus,
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