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Massive Number of Antigen-Specific CD4 T
Cells during Vaccination with Live
Attenuated Salmonella Causes Interclonal
Competition
Aparna Srinivasan, Joseph Foley and Stephen J. McSorley
J Immunol 2004; 172:6884-6893; ;
doi: 10.4049/jimmunol.172.11.6884
http://www.jimmunol.org/content/172/11/6884
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Copyright © 2004 by The American Association of
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References
The Journal of Immunology
Massive Number of Antigen-Specific CD4 T Cells during
Vaccination with Live Attenuated Salmonella Causes
Interclonal Competition1
Aparna Srinivasan, Joseph Foley, and Stephen J. McSorley2
The clonal burst size of CD4 T cells is predicted to be less than that of CD8 T cells. In this study, we demonstrate that massive
numbers of Ag-specific CD4 T cells respond during vaccination of mice with live attenuated Salmonella, reaching a peak frequency
of ⬃50% of CD4 T cells. Salmonella-specific T cells persisted at high frequency for several weeks and could be detected in the
memory population for months after infection. Surprisingly, the expansion of endogenous Salmonella-specific CD4 T cells prevented the persistence of adoptively transferred Salmonella-specific T cells in vivo, demonstrating interclonal competition for
access to the memory compartment. The Journal of Immunology, 2004, 172: 6884 – 6893.
to the notion that the clonal burst size of CD4 T cells is restricted,
perhaps due to inefficient Ag presentation of class-II restricted
epitopes, or an intrinsic defect in the proliferative program of CD4
cells (11). Indeed, studies using an adoptive transfer model where
Listeria-specific TCR-transgenic CD4 and CD8 cells could be directly visualized in vivo noted a clear difference in the proliferative
capacity of these two populations (21). However, it is not clear
whether this represents a general phenomenon for all CD4 T cells
or is only true in models of infection where CD8 effector cells are
critical for pathogen clearance.
The resolution of Salmonella enterica serovar typhimurium
(hereafter referred to as Salmonella typhimurium) infection is critically dependent upon the activation of CD4 T cells and the production of IFN-␥ to activate infected macrophages (22, 23).
Furthermore, live vaccine strains of Salmonella induce robust
CD4-dependent protective immunity in susceptible mice (24). We
reasoned that this might be a more attractive model to examine the
clonal burst size of CD4 T cells in response to infection.
In this study, we report that a massive number of endogenous
Salmonella-specific CD4 T cells are expanded following vaccination with live attenuated Salmonella, reaching a peak at around
50% of all CD4 T cells. Salmonella-specific CD4 cells are maintained at this high frequency until the bacteria are eliminated and
can be detected in the CD4 memory pool for months after vaccination. We also demonstrate that the massive number of endogenous Salmonella-specific CD4 T cells hinders the survival of Salmonella flagellin-specific CD4 T cells, demonstrating interclonal
competition for access to the memory CD4 compartment.
Materials and Methods
Department of Medicine, Division of Immunology, University of Connecticut Health
Center, Farmington, CT 06030
Mouse strains
Received for publication January 23, 2004. Accepted for publication March 19, 2004.
SM1 Rag-deficient TCR-transgenic mice expressing the Thy1.1 or CD45.1
allele were produced by backcrossing the original C57BL/6 SM1 Ragdeficient line (25) to B6.PL-thy1a/Cy or B6.SJL-PtprcaPep3b/BoyJ mouse
strains (The Jackson Laboratory, Bar Harbor, ME). OT-II TCR-transgenic
mice (26) were generously provided by Dr. L. Lefrancois (University of
Connecticut Health Center). C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and used at 8 –16 wk of age. All
mice were housed in specific pathogen-free conditions and cared for in
accordance with the University of Connecticut Health Center and National
Institutes of Health guidelines.
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 grants from National Institutes of Health (AI056172
and AI055743 to S.J.M) and the Robert Leet and Clara Guthrie Patterson Trust (to
S.J.M.).
2
Address correspondence and reprint requests to Dr. Stephen J. McSorley, Department of
Medicine, Division of Immunology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030. E-mail address: [email protected]
Copyright © 2004 by The American Association of Immunologists, Inc.
0022-1767/04/$02.00
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T
he theory of clonal selection (1) predicts a low frequency
of Ag-specific T cell clones, and this has been confirmed
experimentally (2, 3). A rapidly dividing pathogen could
potentially exploit such an inherent frailty in the adaptive immune
response. Therefore, a successful immune response depends upon
the rapid expansion of pathogen-specific clones that express appropriately rearranged Ag receptors. After pathogen clearance, the
maintenance of these clones at an elevated frequency constitutes
an important aspect of immunological memory (4).
Extensive proliferation of Ag-specific CD8 clones has been reported following viral infection, comprising 50 –70% of all CD8
cells at the peak of the response (5, 6). Contraction of the virusspecific response then follows, leaving an elevated frequency of
Ag-specific cells in the memory pool. The burst size of a given
virus-specific population correlates with frequency in the memory
pool, suggesting that there is minimal interclonal competition between
responding CD8 T cells (5). Some studies have suggested that competition between CD8 clones of different Ag specificity can occur in
vivo (7–9), while others support the idea that competition primarily
occurs between cells of identical specificity (6, 10).
Although a great deal has been learned about the nature of the
CD8 response to viral infection, it is not clear whether the same
rules apply to pathogen-specific CD4 T cells (11). Numerous studies have reported that the frequency of Ag-specific CD8 T cells
substantially exceeds that of Ag-specific CD4 T cells at the peak of
the immune response (12–15). Experiments using in vitro restimulation assays have estimated that around 2–3% of CD4 cells are Ag
specific at the peak of the response to viral infection (12, 16 –19),
although studies with lymphocytic choriomeningitis virus have
suggested it could be as high as 10% (13, 20). These data have led
The Journal of Immunology
Adoptive transfer of SM1 T cells
Spleen and lymph node cells (inguinal, axillary, brachial, cervical, mesenteric, periaortic) were harvested from SM1 RAG-deficient, CD90.1 congenic (or CD45.1 congenic) TCR-transgenic mice, and a pooled single-cell
suspension was generated. A small aliquot of cells was stained using Abs
to CD4, CD90.1 (or CD45.1), and V␤2 (BD Biosciences, San Diego, CA)
and the percentage of SM1 T cells was determined by flow cytometry using
a FACSCalibur (BD Biosciences). Cell numbers were adjusted accordingly
and 2–5 ⫻ 106 SM1 T cells were injected i.v. into recipient C57BL/6 or
OT-II mice. In most experiments, SM1 cells were also stained with CFSE
(27) before adoptive transfer.
Vaccination with live attenuated Salmonella or heat-killed S.
typhimurium (HKST)3
immunity. It is possible that the CD4 burst size is far greater in
models where CD4 T cells are the primary effector cell mediating
pathogen clearance.
Vaccination of C57BL/6 mice with attenuated S. typhimurium
provides protective immunity to challenge with virulent bacteria
that is dependent upon CD4 T cells (24). In initial experiments, we
noted that the total number of splenocytes increased markedly 1
wk after vaccination with attenuated Salmonella, largely due to an
increase in the number of CD11b- and Gr1-expressing non-T cells
(Fig. 1A and data not shown). Although this splenomegaly decreased the percentage of CD4 and CD8 T cells (Fig. 1B), the
absolute number of splenic CD4 and CD8 T cells actually increased ⬃2-fold in the infected spleen (Fig. 1C). Phenotypic analysis of the CD4 population demonstrated that this 2-fold increase
was due to a large increase in the number of cells with an activated
phenotype, CD4⫹CD11ahighCD44⫹ (Fig. 1D and data not shown).
Furthermore, a sizable percentage of CD4 T cells from vaccinated
mice produced IFN-␥ and TNF-␣ directly ex vivo (Fig. 1D). CD4
cells from vaccinated mice did not produce IL-4 or IL-10 (data not
shown), although a small proportion produced IL-2 (Fig. 1D). All
cytokine production by CD4 cells was confined to the
CD4⫹CD11ahigh population (Fig. 1E). A number of non-CD4,
Flow cytometric analysis
Spleen or lymph node cells were incubated on ice for 20 – 45 min in Fc
block (spent culture supernatant from the 24G2 hybridoma, 2% rat serum,
2% mouse serum, and 0.01% sodium azide) in the presence of the relevant
primary Abs. FITC-, PE-, CyChrome-, PE-Cy5-, allophycocyanin-, or biotin-conjugated Abs specific for CD4, CD11a, CD45.1, CD69, CD90.1,
IL-2, IL-4, IL-10, TNF-␣, IFN-␥, V␤2, 3, 4, 5.1/5.2, 6, 7, 8.1/8.2, 8.3, 9,
10b, 11, 12, 13, 14, 17a, and isotype control Abs were purchased from BD
PharMingen (San Diego, CA). Following staining, cells were analyzed by
flow cytometry using a FACSCalibur. Data were analyzed using FlowJo
software (TreeStar, San Carlos, CA).
Tracking SM1 T cells and Salmonella
At various times after infection, spleen or lymph node cells were harvested
in Eagle’s Hanks’ amino acids medium (Biofluids, Rockville, MD) containing 2% FBS and 5 mM EDTA. Serial dilutions of each sample were
plated onto MacConkey agar plates (Difco, Detroit, MI) at 37°C to determine bacterial colonization in each tissue. Cells from the same organ (5 ⫻
106/tube) were then processed and stained as described above.
In vivo cytokine stimulation and intracellular staining
For intracellular cytokine experiments examining SM1 T cells, mice were
injected i.v. with 200 ␮g of flagellin peptide 427– 441 (VQNRFNSAIT
NLGNT) (29) plus 25 ␮g of LPS (Sigma-Aldrich, St. Louis, MO). For
intracellular cytokine experiments examining endogenous Salmonella-specific CD4 T cells, mice were injected i.v. with 1 ⫻ 108 HKST. To examine
cytokine production directly ex vivo, spleens from such injected mice were
harvested at 2 h after injection. Spleen cells were rapidly surface stained,
fixed with formaldehyde, permeabilized using saponin (Sigma-Aldrich),
and stained intracellularly using the Abs described above.
Results
Vaccination with live Salmonella increases the number of CD4
memory cells in the spleen
Although a number of studies have examined the clonal burst size
of CD4 cells (12–21), these have used viral or bacterial models
where CD8 T cell expansion is likely to be required for protective
3
Abbreviations used in this paper: HKST, heat-killed Salmonella typhimurium;
HKLM, heat-killed Listeria monocytogene; SAg, superantigen.
FIGURE 1. Vaccination with live Salmonella causes a large expansion
of activated CD4 T cells in vivo. C57BL/6 mice were vaccinated with 5 ⫻
105 live attenuated Salmonella (vaccinated) or were left untreated (naive).
One week later, spleen cells were stained and the total cell number (A),
percentage of CD4 cells (B), total number of CD4 cells (C) were determined. Data show the mean ⫾ SD of three mice per group and are representative of four individual experiments. D, The surface expression of
CD11a and the production of intracellular cytokines were examined in
CD4 cells from the spleen of naive and vaccinated mice. Data show representative plots of spleen cells from at least three individual mice. Numbers indicate the percentage of spleen cells within the boxed gate. E,
CD11a and intracellular staining after gating on live CD4 T cells from
naive or infected mice.
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Attenuated (AroA⫺D⫺) S. typhimurium (BRD509) (28) was provided by
Dr. D. Xu (University of Glasgow, Glasgow, U.K.). Flagellin-deficient S.
typhimurium (BC490) was generously provided by Dr. B. Cookson (University of Washington, Seattle, WA). Bacteria were routinely grown overnight in Luria-Bertani broth without shaking, and diluted in PBS after an
estimation of bacterial concentration using a spectrophotometer. Aliquots
of live bacterial suspensions were incubated at 75°C for 1 h to generate
Heat-killed S. typhimurium (HKST). Live attenuated (5 ⫻ 105) and heatkilled bacteria (1 ⫻ 108), were administered i.v. into the lateral tail vein of
mice. In all experiments, the live bacterial dose was confirmed by serial
plating onto MacConkey agar plates. In preliminary experiments, the challenge doses chosen for this study were found to generate similar peak SM1
clonal expansion and were therefore chosen for further study. In some
experiments, the recall response of SM1 T cells to flagellin peptide immunization was examined in immunized mice. Adoptively transferred and
previously immunized mice were injected i.v. with 200 ␮g flagellin peptide
and 25 ␮g LPS and clonal expansion of SM1 T cells was examined 3 days
later.
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FIGURE 2. Kinetics of CD4 CD11a⫹ and IFN-␥⫹ expansion after vaccination with attenuated Salmonella. C57BL/6 mice were vaccinated with
5 ⫻ 105 live attenuated Salmonella and the number of CD4 cells,
CD11a⫹CD4 cells, and IFN-␥⫹CD4 cells (A) or number of viable Salmonella (B) determined in the spleen at various time points later. C, CD11apositive and IFN-␥- positive CD4 cells expressed as a percentage of all
CD4 cells in the spleen of infected mice. Data show the mean ⫾ SD of two
to four mice per time point.
Most CD4⫹CD11ahigh T cells in the spleen are Salmonella
specific
These data suggested that the Salmonella burden in the spleen
could be directly responsible for the specific activation of a large
number of CD4 T cells to produce IFN-␥ ex vivo. However, even
if this were true, it still would not account for the vast majority of
the expanded CD4⫹CD11ahigh population found in the spleen after
live vaccination. IFN-␥-producing CD4 cells represent ⬍20% of
the total CD4⫹CD11ahigh population during the response to live
vaccination (Fig. 2C). From these data, it was not possible to distinguish whether non-IFN-␥-producing CD4⫹CD11ahigh cells
represented Salmonella-specific CD4 T cells that had not been recently activated in vivo, or were simply CD4⫹CD11high T cells
that were not specific for Salmonella.
To discriminate between these possibilities, we modified an in
vivo peptide pulse assay that has been used to examine the cytokine production of TCR-transgenic CD4 T cells in vivo (30, 31).
This assay typically involves the i.v. injection of peptide to synchronously activate TCR-transgenic T cells in vivo, followed by
analysis of intracellular cytokine production ex vivo, only 2 h later.
Although the Ag specificity of the endogenous CD4 response to
Salmonella is unknown, we hypothesized that most target Ags
would be present in a HKST Ag preparation. C57BL/6 mice were
therefore vaccinated with live attenuated Salmonella and, 1 wk
later, some mice were injected with HKST. Two hours after this
HKST pulse, spleens were harvested and rapidly processed to examine intracellular cytokine production.
As in our previous experiments, an increased frequency of
IFN-␥- and TNF-␣-producing CD4 T cells was detected in vaccinated mice (Fig. 3A, naive vs vaccinated). These cytokine-producing cells were only found in the CD4⫹CD11ahigh population (Fig.
3B). A very small increase in CD4 IFN-␥ and TNF-␣ production
was detected in naive C57BL/6 mice that had received the HKST
pulse (Fig. 3A, naive ⫹ HKST), and these cells also expressed
CD11ahigh. It is possible that these cells are cross-reactive CD4
memory cells specific for the endogenous bacterial flora, but we
have not examined this hypothesis in detail. A massive increase in
the fraction of CD4 T cells producing IFN-␥ and the amount of
IFN-␥ per cell was detected in vaccinated mice that had been
pulsed with HKST (Fig. 3A, vaccinated ⫹ HKST). Again, this
cytokine response only occurred in the CD11ahigh population and
actually accounted for the vast majority of all CD4⫹CD11ahigh T
cells (Fig. 3B).
It seemed likely that the HKST pulse assay allowed for the
identification of Salmonella-specific T cells and that these cells
accounted for a surprisingly high fraction of the CD4 population,
in mice that were vaccinated with live Salmonella. However, it
was possible that the response to the HKST pulse was either a
nonspecific inflammatory response or a specific response to an
unknown Salmonella superantigen (SAg). To address the latter
concern, we analyzed the TCR V␤ repertoire of CD4 T cells producing IFN-␥ and those not producing IFN-␥ after the HKST
pulse, as SAg responses involve the restricted activation of certain
V␤-expressing T cells (32). Although a 2-fold increase in V␤9-,
12-, and 17a-expressing CD4 T cells was noted among IFN-␥positive cells, there was no obvious bias in the V␤ repertoire that
would indicate a SAg response (Fig. 3C). To examine the effect of
nonspecific bacterial inflammation on CD4 IFN-␥ production, we
pulsed vaccinated mice with HKST or heat-killed Listeria monocytogenes (HKLM). Although HKST induced CD4 IFN-␥ production, pulsing vaccinated mice with HKLM did not increase the
frequency of IFN-␥-producing CD4 cells detected in the spleen
(Fig. 3D and data not shown). We conclude that the in vivo pulse
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non-CD8 cells in vaccinated mice stained positive with all Abs,
including isotype control Abs (Fig. 1D). This nonspecific staining
is likely to result from the increased number of CD11b⫹ cells in
the spleen of vaccinated mice.
We examined the in vivo kinetics of the CD4⫹CD11ahigh response. Vaccination with live Salmonella caused a transient increase in total splenic CD4 numbers that had largely dissipated 2
mo later (Fig. 2A, total CD4 cells). The increase in CD4 numbers
was due to a large expansion of the CD4⫹CD11ahigh T cell population (Fig. 2A, total CD11a⫹ CD4 cells). This kinetics of the
CD4⫹CD11ahigh response had three distinct phases: 1) an expansion phase between day 0 and days 7–14, when the absolute number of these cells increased ⬃10-fold; 2) a plateau until days 21–
28, when the number of these cells remained relatively constant at
a high level; 3) a contraction phase after days 21–28, when the
number of cells declined steadily (Fig. 2A).
We also examined the kinetics of the CD4⫹IFN-␥⫹ response,
the magnitude and kinetics of which was slightly different (Fig. 2A,
total IFN-␥⫹ CD4 cells). An increased frequency of CD4⫹IFN␥⫹-producing cells was detected as early as 3 days after live vaccination. This response peaked at around 1 wk and stayed relatively constant until 3 wk following vaccination, after which it
disappeared completely. Interestingly, the kinetics of the IFN-␥producing CD4 cell response closely mirrored the kinetics of the
bacterial burden in the spleen (Fig. 2B). Numbers of Salmonella
increased during the first week, stayed at relatively high levels for
2–3 wk, and then rapidly disappeared.
CD4 T CELL RESPONSE TO LIVE ATTENUATED Salmonella
The Journal of Immunology
6887
assay allows the detection of endogenous Salmonella-specific CD4
T cells in vivo and that these cells comprise a surprisingly large
fraction of the CD4 population in vaccinated mice.
Live vaccination causes a massive increase in
Salmonella-specific CD4 T cells but vaccination with HKST
does not
We used the HKST pulse assay to examine the frequency and
kinetics of the endogenous Salmonella-specific CD4 response.
These data demonstrate that a huge clonal expansion of Salmonella-specific CD4 T cells occurred in the first week after live vac-
cination (Fig. 4A, total IFN-␥⫹ CD4 cells). The number of these
cells persisted at a very high level for 3– 4 wk, after which there
was a significant decline. Importantly, the kinetics of IFN-␥-producing cells closely mirrored the kinetics of the CD11a⫹ population in response to vaccination (Fig. 4A). Indeed, it was possible to
account for all of the increase in the CD4⫹CD11ahigh population as
being due to an increase in Salmonella-specific CD4 T cells (Fig.
4B). At the peak of the response, Salmonella-specific CD4 T cells
accounted for ⬃50% of all CD4 T cells (Fig. 4B). Among Salmonella-specific CD4 cells, ⬃30 – 40% also produced TNF-␣ (Fig.
4B) and ⬃5% produced IL-2 (data not shown). No production of
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FIGURE 3. Pulsing infected mice with HKST allows the identification of endogenous Salmonella-specific CD4 T cells in vivo. A, Groups of C57BL/6 mice
were vaccinated with 5 ⫻ 105 live attenuated Salmonella and 1 wk later some mice were injected i.v. with 1 ⫻ 108 HKST. Spleens were harvested 2 h later and
intracellular production of IFN-␥ and TNF-␣ by CD4 T cells was determined. Data show cytokine production among live spleen cells and are representative of
two to three mice per group and four individual experiments. Numbers represent the percentage of spleen cells falling within the boxed gate. B, CD11a expression
and cytokine production from the same groups after gating on CD4 T cells. C, Groups of C57BL/6 mice were vaccinated with 5 ⫻ 105 live attenuated Salmonella
and 3 wk later injected i.v. with 1 ⫻ 108 HKST. Spleens were harvested 2 h later and intracellular production of IFN-␥ and expression of TCR V␤ receptors were
determined among CD4 T cells. IFN-␥-negative and -positive cells were defined using a boxed gate similar to that shown in A. Data show the percentage of CD4
cells expressing a particular V␤ chain after gating on IFN-␥-positive or -negative CD4 cells. D, Groups of C57BL/6 mice were vaccinated with 5 ⫻ 105 live
attenuated Salmonella and 1 wk later some mice were injected i.v. with 1 ⫻ 108 HKST or 1 ⫻ 108 HKLM. Spleens were harvested 2 h later and intracellular
production of IFN-␥ was determined. Numbers represent the percentage of spleen cells falling within the boxed gate.
6888
nodes and axillary-brachial-linguinal (ABI) nodes of C57BL/6
mice after adoptive transfer (Fig. 5). In response to live vaccination, SM1 T cells expanded in the spleen but not in the mesenteric
lymph nodes or peripheral ABI lymph nodes (Fig. 5). This restricted activation of SM1 T cells is reminiscent of our previous
observation that SM1 T cell expansion is limited to the gutassociated lymphoid tissue following oral Salmonella infection
(25) and likely reflects an intrinsic property of the immune response to Salmonella flagellin. However, the most surprising
finding was that by 10 days after vaccination very few SM1 T cells
could be detected in the spleen or any other lymphoid tissue
examined (Fig. 5). Therefore, despite the massive clonal expansion
of adoptively transferred SM1 T cells at days 3–5, they are not
found at an elevated frequency at later time points. Indeed, by day
10, frequencies of SM1 T cells typically drop below the low level
detected in transfer only mice (Fig. 5).
It was possible that the unusual inability to generate SM1 memory cells was an intrinsic property of SM1 T cells or of flagellinspecific T cells in general. We therefore attempted to generate
memory SM1 T cells after immunization with HKST or flagellin
peptide and LPS. After immunization with HKST or peptide/LPS,
SM1 T cells expanded in the spleen, mesenteric lymph nodes, and
ABI lymph nodes (Fig. 5 and data not shown). By day 10 or 20
after immunization, an increased frequency of SM1 T cells was
detected in all lymphoid tissues analyzed (Fig. 5 and data not
shown). In some experiments, the peak expansion of SM1 cells to
HKST was greater than that of live vaccination (Fig. 5B). However, this did not explain the ability of these cells to persist in vivo.
The relative decrease from the peak frequency was always severalfold greater following live vaccination than after HKST vaccination. Thus, the inability to generate an elevated frequency of
IL-4 or IL-10 by CD4 T cells was detected at any time point using
this assay (data not shown).
We examined whether this expansion of Salmonella-specific
CD4 cells could be observed after vaccination with Salmonella
Ags or whether it only occurred following vaccination with live
bacteria. C57BL/6 mice were immunized i.v. with HKST and 6
and 11 days later, IFN-␥ and TNF-␣ production of CD4 T cells
was examined using the HKST pulse assay exactly as described
above. CD4 T cells in the spleen of HKST-vaccinated mice did not
produce large quantities of cytokines in response to the in vivo
pulse (Fig. 4C and data not shown)
Salmonella-specific TCR-transgenic cells do not develop into
memory cells following vaccination with live Salmonella
We sought to examine the clonal expansion and development of
Salmonella-specific memory CD4 cells in more detail using an
adoptive transfer approach. We previously generated the SM1
TCR-transgenic mouse specific for Salmonella flagellin (25), and
therefore decided to use this model to examine the CD4 response to live vaccination. A small population of SM1 T cells
(CD4⫹CD90.1⫹) was detected in the spleen, mesenteric lymph
FIGURE 5. SM1 T cells do not persist after live attenuated Salmonella
vaccination. C57BL/6 mice were adoptively transferred with 2 ⫻ 106 SM1
T cells and groups of mice were vaccinated the following day with 5 ⫻ 105
live attenuated Salmonella or 1 ⫻ 108 HKST. A, Spleen cells were harvested 5 and 10 days later and the percentage of SM1 T cells was determined. Plots are representative of three mice per group and three similar
experiments. B, In another similar experiment, spleen, mesenteric lymph
nodes, and ABI lymph nodes were harvested at various time points following vaccination with live Salmonella or HKST and the percentage of
SM1 T cells in each tissue was determined by flow cytometry.
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FIGURE 4. Massive expansion of Salmonella-specific CD4 T cells after
vaccination with live Salmonella but not HKST. A, C57BL/6 mice were
vaccinated with 5 ⫻ 105 live attenuated Salmonella and injected with 1 ⫻
108 HKST at various time points later. Two hours after HKST injection,
spleen cells were harvested and the number of CD4 cells, CD11a⫹ CD4
cells, and IFN-␥⫹ CD4 cells was determined. Data show the mean ⫾ SD
of two to four mice per time point. B, CD11a-positive and IFN-␥-positive
CD4 cells expressed as a percentage of all CD4 cells in the spleen of
infected mice. C, C57BL/6 mice were vaccinated with 1 ⫻ 108 HKST and
11 days later some mice were injected with 1 ⫻ 108 HKST to stimulate
cytokine production. Two hours later, spleen cells were harvested and cytokine production of CD4 T cells was examined by intracellular staining.
The number of CD4 cells, CD11a⫹ CD4 cells, and IFN-␥⫹ CD4 cells was
determined. Numbers indicate the percentage of spleen cells within the
boxed gate.
CD4 T CELL RESPONSE TO LIVE ATTENUATED Salmonella
The Journal of Immunology
6889
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FIGURE 6. SM1 T cells are functionally responsive to peptide in mice administered live Salmonella. C57BL/6 mice were adoptively transferred with
2 ⫻ 106 CFSE-labeled SM1 T cells and groups of mice were vaccinated the following day with 5 ⫻ 105 live attenuated Salmonella or 1 ⫻ 108 HKST.
A, Spleen cells were harvested 5 days later and CFSE staining was examined by flow cytometry after gating on CD4⫹CD90.1⫹ SM1 T cells. Data are
representative of three individual mice and two separate experiments. B, C57BL/6 mice were adoptively transferred with 2 ⫻ 106 CFSE-labeled SM1 T
cells and groups of mice were vaccinated the following day with 5 ⫻ 105 live attenuated Salmonella or 1 ⫻ 108 HKST. Five days after vaccination, some
mice were injected i.v. with 200 ␮g of flagellin peptide and 25 ␮g of LPS (⫹ peptide), and some mice were left untreated (⫺ peptide). Two hours after
peptide injection, spleen cells were harvested and stained using various different Abs. Data show surface staining for CD69 and intracellular staining for
IL-2 or IFN-␥ after gating on splenic CD4⫹CD90.1⫹ SM1 T cells. Plots from individual mice are shown and are representative of two individual mice and
two individual experiments. Numbers represent the percentage of SM1 T cells in the boxed gate. C, C57BL/6 mice were adoptively transferred with 2 ⫻
106 SM1 T cells and groups of mice were vaccinated the following day with 5 ⫻ 105 live attenuated Salmonella or 1 ⫻ 108 HKST. Two weeks later, groups
of mice were injected i.v. with 200 ␮g of flagellin peptide and LPS. Three days later, spleen cells were harvested and the percentage of SM1 T cells was
determined. Numbers represent the percentage of SM1 T cells in the spleen, as defined by the boxed gate.
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CD4 T CELL RESPONSE TO LIVE ATTENUATED Salmonella
SM1 T cells is a specific property of live vaccination and is not due
to an intrinsic defect in SM1 TCR-transgenic cells.
Vaccination with live Salmonella or HKST induces SM1 T cell
populations with similar phenotypic and functional properties
Endogenous Salmonella-specific T cells prevent the development
of SM1 memory cells
Our data suggested that a massive expansion and maintenance of
endogenous Salmonella-specific T cells occurs in response to live
FIGURE 7. Salmonella-specific endogenous CD4 T cells hinder the
persistence of SM1 T cells in vivo. A, C57BL/6 or OT-II mice were adoptively transferred with CFSE-labeled 2 ⫻ 106 SM1 T cells and groups of
mice were vaccinated the following day with 5 ⫻ 105 live attenuated Salmonella. Spleen cells were harvested 3 and 10 days later, and the percentage of SM1 T cells was determined. CFSE plots are gated only on SM1 T
cells, as defined by the boxed gate shown. Numbers represent the percentage of SM1 T cells in the spleen. B, Groups of C57BL/6 mice were vaccinated with 5 ⫻ 105 live attenuated Salmonella and 1 wk later some mice
were injected i.v. with 1 ⫻ 108 HKST prepared from flagellated (HKST)
or aflagellate bacteria (aflagellate HKST). Spleens were harvested 2 h later
and intracellular production of IFN-␥ was determined. Data show the percentage of CD4 T cells producing IFN-␥ as a percentage of all CD4 T cells
in the spleen.
vaccination. In contrast, SM1 T cells expand but are unable to
survive at later time points. We therefore examined the possibility
that these two populations of cells compete with each other during
live vaccination. We decided to examine SM1 T cell persistence in
a model where endogenous CD4 T cell competition cannot occur
in vivo. SM1 T cells were therefore adoptively transferred into
C57BL/6 mice or OT-II TCR-transgenic mice. All endogenous
CD4 T cells in the periphery of OT-II mice are specific for OVA
and should be unable to respond during vaccination with live Salmonella. Three days following live Salmonella vaccination, bacterial counts in the spleen of both recipient groups were similar
(data not shown). SM1 T cells in both C57BL/6 and OT-II recipients expanded in response to vaccination, increased expression of
CD11a, and underwent multiple rounds of cell division, as observed by CFSE staining (Fig. 7A, day 3). SM1 T cells in OT-II
mice expanded slightly more than those in C57BL/6 mice. As in
previous experiments, SM1 T cells did not persist in the spleen of
C57BL/6 mice vaccinated with live Salmonella (Fig. 7A, day 10).
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Immunizing mice with live Salmonella or HKST generated a similar frequency of splenic SM1 cells on day 5, but a very different
frequency by day 10. To understand this difference, we examined
the phenotypic and functional properties of SM1 T cells at day 5.
Analysis of later time points was difficult due to the rapid loss of
the SM1 population in mice vaccinated with live Salmonella (Fig.
5). On day 5, SM1 cells in the spleen of both groups of mice had
undergone multiple rounds of cell division and increased surface
expression of CD11a (Fig. 6A) and CD44 (data not shown).
Recent reports suggest that IFN-␥-producing CD4 T cells may
be short-lived and unable to develop into memory T cells (33). It
was possible that live Salmonella induce a more pronounced
IFN-␥ response in SM1 T cells, leading to their inability to survive
at later time points. We therefore examined the cytokine production of SM1 T cells directly ex vivo after vaccination with live
Salmonella or HKST. Five days after vaccination, SM1 T cells
were uniformly CD69low, IL-2⫺, and IFN-␥⫺ (Fig. 6B). We next
sought to examine the functional response of these SM1 T cells
after in vivo stimulation with flagellin peptide and LPS. Five days
after vaccination, transfer only, live Salmonella, or HKST mice
were injected i.v. with peptide/LPS to activate SM1 cells. Intracellular cytokine production was examined 2 h later. SM1 T cells
in transfer only, live Salmonella, or HKST mice increased surface
expression of CD69 in response to in vivo peptide stimulation,
indicating that all of these cells encountered peptide (Fig. 6B).
Approximately 20% of SM1 T cells transfer-only mice produced
IL-2 in response to peptide stimulation, and this was increased in
mice exposed to live Salmonella or HKST (Fig. 6B). Very few
naive SM1 cells in transfer-only mice produced IFN-␥ in response
to peptide stimulation (Fig. 6B). A higher percentage of SM1 cells
produced IFN-␥ in mice administered live Salmonella or HKST,
but there was no noticeable difference between the vaccination
regimens (Fig. 6B). These data indicate that SM1 T cells in mice
immunized with HKST or live Salmonella are functionally similar
and that excess IFN-␥ production is unlikely to explain the loss of
SM1 T cells observed at later time points.
Next, we examined whether the few SM1 T cells that remain at
later time points in mice vaccinated with live Salmonella are actually functionally responsive. C57BL/6 mice were adoptively
transferred with SM1 T cells and vaccinated with HKST or live
Salmonella or were left unimmunized (transfer only). Two weeks
later, some mice were immunized i.v. with flagellin peptide/LPS
and clonal expansion of SM1 T cells was examined 3 days later. A
small population of SM1 T cells was detected in the spleen of
transfer-only mice, and these cells expanded in response to peptide/LPS stimulation (Fig. 6C). In agreement with previous experiments, HKST-immunized mice had a higher frequency of SM1 T
cells in the spleen than transfer-only mice and these cells also
expanded in response to peptide/LPS challenge (Fig. 6C). Although live Salmonella-vaccinated mice had a very low frequency
of SM1 T cells in the spleen, these cells expanded after exposure
to peptide/LPS. Therefore, the few SM1 T cells remaining in the
spleen of mice administered live Salmonella are functionally
responsive.
The Journal of Immunology
In this particular experiment, SM1 T cells had not fully diluted
CFSE in response to live Salmonella, allowing clear visualization
of divided and undivided SM1 cells. The failure of SM1 T cells to
persist at day 10 corresponded with the preferential loss of the
most CFSE-dilute cells (Fig. 7A, C57BL/6, day 3 vs day 10). In
contrast, SM1 T cells persisted in OT-II recipient mice at day 10,
and this included the survival of the most CFSE-dilute cells (Fig.
7A, OT-II, day 3 vs day 10). We conclude that SM1 T cells fail to
persist due to competition from the endogenous Salmonella-specific CD4 T cell response to live Salmonella.
Flagellin is not a major target Ag of endogenous
Salmonella-specific CD4 T cells
Discussion
We have analyzed the response of CD4 T cells to vaccination with
live attenuated S. typhimurium. Our data demonstrate that a large
increase in the number of CD4⫹CD11ahigh T cells occurs rapidly
after vaccination and is maintained for several weeks. A rise in the
number of CD4⫹CD44high cells has also been reported in resistant
mice after infection with virulent Salmonella (36). Together these
data suggest that a large expansion of CD4 cells with an activated
phenotype is typical of the immune response to Salmonella.
Our in vivo HKST pulse assay demonstrates that the vast majority of these CD4⫹CD11ahigh cells are Salmonella specific. The
2-h stimulation period of the HKST pulse assay ensures that this
assay measures effector cytokine production from previously expanded CD4 cells and not the clonal expansion of CD4 T cells in
response to HKST.
It is possible that the IFN-␥ production detected using the HKST
pulse assay does not derive from Salmonella-specific CD4 T cells.
However, we think this is unlikely for the following reasons: First,
cytokine production was not detected in the naive CD11alow population and comes solely from cells with an activated CD11ahigh
surface phenotype. Therefore, if this assay measures a nonspecific
response, it is a nonspecific response that is peculiar to CD4 T cells
with an Ag-experienced surface phenotype. Second, injection of
HKLM did not allow the detection of CD4⫹IFN-␥⫹ T cells in the
spleen. Therefore, if this is a nonspecific response to bacterial inflammation, it is one that appears to be particular to Salmonella Ags.
Third, we detected no obvious bias in the CD4 TCR V␤ repertoire of
CD4 T cells producing IFN-␥ in the pulse assay, rendering the possibility of a Salmonella SAg in the HKST preparation unlikely.
Fourth, there is an expansion of the IFN-␥ response in the first few
days after infection and a contraction of this response when bacteria
are cleared. Therefore, the kinetics of IFN-␥ production by the
CD4⫹CD11ahigh population represent the kinetics of an adaptive
immune response rather than an innate response to inflammation.
Lastly, a high frequency of CD4⫹CD11a⫹ cells producing IFN-␥
was detected after the complete clearance of attenuated bacteria.
Therefore, we can detect immune memory, a hallmark of adaptive
immune responses. Taken together, these data support the conclusion that the HKST pulse assay detects Salmonella-specific CD4 T
cells in vivo. Although we think the evidence is strong that the
assay detects Ag-specific CD4 T cells, we have not conclusively
demonstrated that ligation of the TCR is required during the recall
response. Therefore, it remains possible that a large number of
Salmonella-specific CD4 T cells expand after vaccination but that
these cells acquire the ability to respond in an Ag-nonspecific manner to the HKST pulse. Whatever the exact mechanism, the detection of a response months after infection indicates that our assay
allows the detection of an adaptive immune response to
Salmonella.
We have therefore been able to detect a surprisingly large population of Ag-specific CD4 T cells in response to live vaccination.
At the peak of the response, this comprises ⬃50% of the entire
CD4 population in the spleen and in some experiments can be as
high as 65% (data not shown). The detection of an Ag-specific
CD4 T cell response of this magnitude is unprecedented. A number
of reports have measured the CD4 burst size in response to infection to be ⬃2–3% of all CD4 T cells (12, 16 –19), although one
report measured the peak CD4 T cell response to lymphocytic
choriomeningitis virus at ⬃10% of CD4 T cells (13).
This raises the question of why the Salmonella-specific CD4
response would be so large in response to live vaccination? A
simple explanation is that previous studies have examined CD4 T
cell responses under conditions where effector CD4 expansion is
not absolutely critical for the development of protective immunity.
During viral (12, 16 –19) or Listeria infection (21), although CD4
T cells can clearly contribute to the antiviral response (37), the
expansion of CD8 effector cells is more likely to be critical for
viral clearance. In contrast, CD4 T cells are absolutely required for
the clearance of attenuated Salmonella strains in vivo (23, 38).
Analysis of the CD4 clonal burst size in other infectious disease
models may therefore demonstrate much higher frequencies than
those reported for viral infections.
However, we favor an alternative hypothesis. We think it possible that the huge expansion of Salmonella-specific CD4 T cells
reported here is peculiar to this infectious disease model. This
might occur because of the presence of an unusually high frequency of endogenous Salmonella-specific T cells in the secondary
lymphoid tissues of naive mice. These cells might arise naturally
due to cross-reactivity between Salmonella epitopes and those
present in the normal bacterial flora. Indeed, our analysis of naive
C57BL/6 mice indicates the presence of a small but visible population of HKST-specific CD4 T cells that produce IFN-␥ (Fig. 3A,
naive ⫹ HKST). Future experiments using mice with defined gut
flora will help clarify whether these cells arose in response to
cross-reactive bacterial epitopes and are responsible for the large
clonal burst in response to Salmonella.
What Ags are recognized by Salmonella-specific CD4 T cells in
vivo? Only two Ags have been clearly defined as targets of Salmonella-specific T cells, flagellin and SipC (29, 35, 39). Our experiments suggest that flagellin is not a major target of CD4 T cells
in vivo, despite its identification as a target of numerous Salmonella-specific T cell clones in vitro (29, 35). It is possible that the
intrinsic adjuvant properties of flagellin (40), mediated through
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It still was not clear why endogenous CD4 T cells would have a
selective survival advantage over SM1 T cells. In fact, previous
adoptive transfer experiments have reported that an increased frequency of TCR-transgenic cells actually blocks the clonal expansion of endogenous T cells (3, 6, 34). Although flagellin has been
identified as a target of Salmonella-specific CD4 T cell clones (29,
35), it remained possible the majority of the endogenous CD4 response was directed at other unknown proteins and that flagellinspecific CD4 T cells do not compete well with these other specificities. We therefore sought to use the HKST pulse assay to
estimate the fraction of endogenous Salmonella-specific CD4 T
cells that are specific for flagellin after live vaccination. HKST was
prepared from flagellate or aflagellate Salmonella and these Ag
preparations compared for their ability to activate endogenous
CD4 T cells in vaccinated mice. Surprisingly, we found that the
endogenous CD4 response to flagellate HKST was roughly equivalent to that of aflagellate HKST (Fig. 7B), suggesting that flagellin
is not a major target of Salmonella-specific CD4 T cells in vivo.
Therefore, it is likely that SM1 T cells do not compete well with
the endogenous Salmonella-specific response due to differences in
the Ag specificity of these two populations.
6891
6892
Acknowledgments
We thank Dr. A. Vella and Dr. L. Lefrancois for helpful discussion.
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