DOMAIN 8 PATHOGENESIS
Adaptive Immune Responses
Received: 21 August 2008
Accepted: 17 November 2008
Posted: 17 September 2009
Supercedes previous posting at EcoSal.org.
Editor: Michael S. Donnenberg, University of
Maryland, School of Medicine, Baltimore, MD
Citation: EcoSal Plus 2013; doi:10.1128/
ecosalplus.8.8.11.
Correspondence: Brad T. Cookson: cookson@u.
washington.edu
Copyright: © 2013 American Society for
Microbiology. All rights reserved.
doi:10.1128/ecosalplus.8.8.11
ASMScience.org/EcoSalPlus
during Salmonella Infection
LISA A. CUMMINGS,1 BROOKE L. DEATHERAGE,2 AND
BRAD T. COOKSON1,2
1
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195
2
Department of Microbiology, University of Washington, Seattle, WA 98195
ABSTRACT The interaction between Salmonella and its host is complex and dynamic: the
host mounts an immune defense against the pathogen, which in turn acts to reduce,
evade, or exploit these responses to successfully colonize the host. Although the exact
mechanisms mediating protective immunity are poorly understood, it is known that T cells
are a critical component of immunity to Salmonella infection, and a robust T-cell response
is required for both clearance of primary infection and resistance to subsequent challenge.
B-cell functions, including but not limited to antibody production, are also required for
generation of protective immunity. Additionally, interactions among host cells are
essential. For example, antigen-presenting cells (including B cells) express cytokines that
participate in CD4+ T cell activation and differentiation. Differentiated CD4+ T cells secrete
cytokines that have both autocrine and paracrine functions, including recruitment and
activation of phagocytes, and stimulation of B cell isotype class switching and affinity
maturation. Multiple bacterium-directed mechanisms, including altered antigen expression
and bioavailability and interference with antigen-presenting cell activation and function,
combine to modify Salmonella’s “pathogenic signature” in order to minimize its susceptibility
to host immune surveillance. Therefore, a more complete understanding of adaptive
immune responses may provide insights into pathogenic bacterial functions. Continued
identification of adaptive immune targets will guide rational vaccine development, provide
insights into host functions required to resist Salmonella infection, and correspondingly
provide valuable reagents for defining the critical pathogenic capabilities of Salmonella
that contribute to their success in causing acute and chronic infections.
INTRODUCTION
The significance of salmonellosis as a medical problem is readily appreci-
ated by noting that vaccination efforts to protect against the disease began
in the latter part of the 19th century (1). Six decades later, murine models
of Salmonella infection, in which Salmonella enterica serovar Typhimurium
infection in mice served as a model for Salmonella enterica serovar Typhi
infection in humans, began to provide important new insights. Greater
protection was afforded by sublethal infection with viable bacteria than by
administration of heat-killed vaccines (2). Protection generated by live
organisms did not correlate with anti-lipopolysaccharide (anti-LPS)
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Cummings et al.

Figure 1 Disease outcome is the cumulative result of Salmonella interactions with host adaptive immune functions. During Salmonella
infection, there are several points at which pathogen and host immune cells interact. Multiple outcomes for each interaction are possible; the
predominant outcome is determined not only by the virulence of the bacterium, but also the innate resistance or susceptibility of the host, and
previous exposure of the host to the pathogen (host immune status). Uptake of bacteria by APCs (A) results in either APC elimination by
pyroptosis (B) or APC survival (C). Pyroptosis leads to release of the inflammatory cytokines IL-1β and IL-18, and possibly releases bacteria or
bacterial Ags for uptake by bystander APCs. Some APCs, such as macrophages, are capable of destroying intracellular bacteria (D). If APCs
survive, they may be able to process and present Ag in the context of MHC (E). Salmonella interferes with this process via multiple mechanisms
including repression of Ag expression, bacterial surface modifications that reduce Ag bioavailability and APC stimulation/maturation, and other
SPI-2 -dependent processes that mediate bacterial survival within the phagosome (F). Protected from antibody detection, intracellular Salmonella
can utilize APCs as vehicles for systemic dissemination and replication (G). If APCs are able to overcome bacterial interference to process and
present Ag to T cells (E), Salmonella may still inhibit T-cell activation via stimulation of nitric oxide (NO) production and other direct,
suppressive effects (H). Recognition of peptide-MHC on APCs by TCR-expressing naïve T cells leads to activation and expansion of Ag-specific
effector T cells (I). Effector CD4+ T cells provide help for the activation of CD8+ CTLs (leading to cytokine production and lysis of infected host
cells [J]), and B cells (K). B cells and T cells work synergistically: T cells provide help for antibody production, isotype class switching, and cytokine
production by B cells, while B-cell cytokine production supports Th-1 T-cell differentiation, and Ig on B-cell surfaces mediate Ag capture for
processing and presentation to T cells (K). Cytokines such as IFN-γ are produced by effector T cells to further enhance APC function and activate

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 Adaptive Immune Responses during Salmonella Infection

antibody responses (2), and passive administration of
antibody prolonged survival but did not afford com-
plete protection. Subsequent studies indicated that T
cells contributed to protective immunity (3, 4, 5). Thus,
the principle that both cellular and humoral responses
were required for protection was established (6). Im-
portantly, this concept was confirmed in 1993 by the
demonstration that adoptive transfer of immune serum
and T cells provided protection to naïve, susceptible
hosts (7).
Immunity requiring both T-cell responses and immu-
noglobulins (Igs) from B cells is consistent with our
understanding of Salmonella as a facultatively intracel-
lular pathogen. Bacteria encounter phagocytes either
within the gastrointestinal tract (8), or during penetra-
tion of the mucosal epithelium (9). Salmonella-specific Ig
produced by B cells can opsonize extracellular bacteria
for phagocytic uptake. Salmonella then spread systemi-
cally within phagocytes (9) to colonize target organs
such as liver and spleen (10, 11). Salmonella is able to
survive and replicate within macrophages and dendritic
cells (DCs) in vivo. Inside these professional antigen-
presenting cells (APCs), major histocompatibility com-
plexes (MHCs) serve as receptors surveying the host
cellular cytosol and vacuolar compartments for the pre-
sence of foreign peptides. When bound by MHC, these
peptides are presented on the APC surface for recogni-
tion by T-cell antigen receptors (TCRs). In addition to
macrophages and DCs, B cells can also act as APCs.
These cells also express MHC, and surface-expressed Ig
promotes high-efficiency antigen (Ag) capture for Ag
presentation. TCR recognition of Ag presented by APCs
leads to activation and expansion of CD4+ T cells. In
addition to Ig production, B cells express cytokines that
appear to participate in the differentiation and function
of effector CD4+ T cells (12). Activated CD4+ T cells in
turn provide critical stimulation for B cells to undergo
expansion and Ig class switching, as well as for activation
and growth of cytotoxic CD8+ T cells and phagocytic
cells such as macrophages (13). Thus, a pathogen whose
virulence requires survival inside host phagocytic cells
(14) provides a contextual framework for understanding
adaptive immunity (Fig. 1).
The generation of protective immunity in the naïve host
relies on the reactive milieu of innate immune responses
elicited at the initiation of bacterial infection by DCs, and
to a lesser extent, macrophages. The inflammatory re-
sponse critically benefits the host by helping to generate
B- and T-cell responses, but recruitment of phagocytic
cells to the site of initial bacterial invasion and replication
may facilitate systemic bacterial spread (15). In addition,
Salmonella are capable of destroying both macrophages
and DCs by an inflammatory process called pyroptosis
(see Chapter Host Cell Death) (16, 17, 18), which
abrogates their important APC function(s) and thereby
antagonizes initiation of the adaptive immune response
(Fig. 1B and 2C). Thus, Salmonella infection readily kills
the naïve susceptible murine host. In contrast, the ob-
servation that Salmonella also causes chronic coloniza-
tion (19, 20), made famous by Typhoid Mary in the last
century, indicates that Salmonella have evolved mecha-
nisms to finely balance these seemingly opposing cellular
reactions.
Despite recent advances, many aspects of salmonellosis
and immunity to Salmonella infection remain enigmatic.
The mechanisms by which virulent Salmonella evades
primary immune responses to mount lethal infections in
naïve hosts are incompletely understood, as are the
means by which live attenuated bacteria generate pro-
tective immunity. The identity of Ags recognized by the
adaptive immune response, and the precise roles of T
cells and B cells in providing immunity are likewise
poorly defined. However, our view is that this system
provides a rich model for defining significant and fun-
damental aspects of the immunopathogenesis of host-
pathogen interactions. Both genetic (21, 22) and acquired
(23) immune defects lead to increased susceptibility to
salmonellosis, which is responsible for the annual loss of
hundreds of thousands of lives (24). Salmonella infec-
tions therefore remain a contemporary scientific problem
of substantial medical and economic significance mea-
sured annually in the billions of dollars worldwide (25,
26). Here, we highlight aspects of adaptive immune re-
sponses to Salmonella that shed critical light on our
current state of understanding and provide a foundation
to help address important questions in the future.

bacterial degradation by macrophages (L). In an immune host, previously primed Ag-specific memory T cells (M) may be activated by APCs that
process and present Ag (N); activation of these cells and their effector functions (O) is much more rapid than for naïve T cells. In addition,
circulating antibodies primed by previous immunization facilitate bacterial uptake via opsonization (P), accelerating the efficiency of Ag
presentation up to 1,000-fold. Thus, the ultimate outcome of infection is the cumulative result of complex interactions between pathogen and
host.

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Cummings et al.

Figure 2 Development of adaptive immune responses during Salmonella infection. Interactions with Salmonella and its host are dynamic and
complex. Flagellated and nonflagellated Salmonella are present in the gut lumen (A), where they must overcome initial barriers including the
glycocalyx layer, antimicrobial peptides, and sIgA. Salmonella cross the epithelium (B) via M cells, by inducing endocytosis in epithelial cells, or
following uptake by CX3CR1+ DCs. Bacteria in the gut lumen (A), within epithelial cells (B) and in the PP (C) express FliC protein; interaction of
FliC with TLR5 initiates an inflammatory response characterized by production of IL-8 and CCL20 by epithelial cells. Release of inflammatory
mediators triggers infiltration of macrophages, neutrophils, and CCR6+ DC. Interaction of these cells with Salmonella results in at least three
possible outcomes: (1) Phagocytosis of bacteria by DC or macrophages, ultimately resulting in inflammatory cell death (flagellin-dependent
pyroptosis). Pyroptosis eliminates potential APCs, leads to release of the inflammatory cytokines IL-1β and IL-18, and possibly releases Ags for
uptake by bystander APCs (note that flagellin-negative bacteria have a reduced ability to trigger inflammation via TLR5 or pyroptosis). (2) Uptake
of bacteria, which persist within the phagocyte. This interaction can lead to production of NO by APCs (inhibitory for T-cell activation) and
upregulation of MHC and costimulatory molecules on DC. In addition, these cells could provide a means of transport to systemic sites such as the
liver and spleen. (3) Bacteria are phagocytosed by neutrophils or macrophages and degraded. Mature CCR6+ DC in the PP (C) process and
present Ags to naïve T cells; Ags acquired for processing and presentation are restricted to those expressed by bacteria in the gut lumen (A) or PP
(C), or Ags that are present in gut lumen, and disassociated from the bacterial soma ([A] MVs, flagellin). Mature DCs that have processed and
presented Ag on surface MHC enter into an “activation feedback” loop with naïve T cells: TNF-α and IL-12 produced by DCs enhance activation
and expansion of Ag-specific T cells, while IFN-γ secretion by activated T cells further stimulates DC function. Memory T cells primed in the PP
(C) express the α4β7-homing receptor as well as CCR6+, predisposing these cells to traffic to the inflamed gut during a secondary infection.

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 Adaptive Immune Responses during Salmonella Infection

ANTIGEN-PRESENTING CELLS
APCs Are Required for the Development of
Ag-Specific T-Cell Responses
The detection of bacterial Ags by T cells and B cells via
their variable receptor molecules (TCR and BCR, re-
spectively) is the cornerstone of adaptive immune rec-
ognition. Unlike B cells, T cells do not recognize bacterial
proteins in their native state. Instead, bacterial Ags must
be processed into peptides and presented by professional
APCs on specialized surface display structures called
MHCs. In general, peptides derived from extracellular
pathogens internalized by the host cell or pathogens
replicating in intracellular vacuoles are displayed on
MHC-II molecules. Correspondingly, peptides derived
from pathogens replicating in the host cell cytosol are
displayed on MHC-I molecules. Presentation on MHC-II
is required for activation of CD4+ T cells, while CD8+ T
cells respond to peptides presented in the context of
MHC-I molecules. Thus, the mammalian host has
evolved mechanisms for immune surveillance of both
extracellular and intracellular pathogens. The importance
of APC function in the development of immunity to
Salmonella infection, and the mechanisms by which Sal-
monella interfere with this function are discussed below.
DCs and Macrophages as Professional APCs
during Salmonella Infection
Because of their ability to process and present Ag to T
cells, professional APCs are critical for immunity to
Salmonella infection. Macrophages and DCs are classified
as professional APCs, although B cells may also serve this
function (reference 27, and see “B-cell functions in ad-
dition to antibody production,” below). Upon Salmonella
encounter in vitro, both macrophages and DCs are
stimulated to upregulate the expression of molecules re-
quired for T-cell activation: costimulatory surface mole-
cules CD40, CD80, and CD86 and immunomodulatory
cytokines gamma interferon (IFN-γ), tumor necrosis
factor alpha (TNF-α), interleukin 12 (IL-12), and IL-18
(28, 29, 30). DCs are well established activators of naïve T
cells (31), and recent advances have begun to reveal a
complex and diverse array of functionally distinct
subpopulations (32, 33, 34). Macrophages are also capa-
ble of priming naïve Salmonella-specific T-cell responses
in vivo (35). Nevertheless, these two cell types may play
different roles in the development of adaptive immunity
(29, 36).
DCs are phagocytic cells, but bacterial uptake induces a
morphological and functional transition from immature
Ag-capturing cells to mature APCs (31). Intracellular
Salmonella persist but do not replicate within bone
marrow-derived DCs (37), and DCs express low levels of
lysosomal proteases, degrading bacterial proteins more
slowly than macrophages. (38). Therefore, the environ-
ment within the mature DC is optimized for Ag
processing and presentation rather than killing bacteria.
DCs reside in the subepithelial area of the Peyer’s patches
(PPs), ideally situated for an initial encounter with in-
vading Salmonella. In contrast, relatively few macro-
phages reside in the PP (39). After oral infection,
colocalization of Salmonella with DCs in the PP and
mesenteric lymph nodes (MLNs) occurs rapidly (39, 40).
In addition, CCR6+ DCs colocalize with Salmonella-
specific T cells in the PP suggesting that DCs are the
primary APCs at early stages of infection (41). DCs in-
fected in vitro are capable of priming Salmonella-specific
CD4+ and CD8+ T-cell responses after adoptive transfer
to naïve mice (30, 42), demonstrating the potential of
DCs to prime naïve T cells in vivo. Indeed, DCs are
absolutely required for activation and expansion of Sal-
monella-specific T cells in the PP (41). Taken together
these findings confirm that DCs play a critical role in the
initiation of adaptive immunity to Salmonella.
Although macrophages are classified as professional
APCs and are capable of presenting Salmonella-derived
Ags to T cells (28, 43, 44, 45), it is unclear whether this is
their primary function during Salmonella infection. Hu-
man macrophages appear to be more efficient in the
production of proinflammatory chemokines (CCl5,
CCL20, and CXCL10) and cytokines (IL-18, TNF-α) than
DCs (29), whereas DCs are more efficient than macro-
phages in stimulating IFN-γ production by CD4+ T cells
(28). This suggests that macrophages may be more

Bacteria within APCs disseminate to MLN (D), where Ag presentation to T cells can also occur. Bacteria in the MLN have undergone complete
adaptations to the intracellular environment: they no longer express FliC, actively reduce Ag bioavailability, and interfere with APC function (see
Fig. 1). Dissemination to systemic sites such as liver and spleen (E) follows MLN colonization. Bacteria replicate within APCs at systemic sites.
However, in naïve hosts, T-cell responses to Ags expressed by intracellular phase bacteria are generally not sufficient in magnitude or quality, or
fail to develop rapidly enough, to combat infection. Further, T cells primed at early stages in the PP (C) will not recognize bacteria growing
intracellularly at systemic sites (E), or that fail to express FliC in the PP (C, left).

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Cummings et al.
important in mediating proinflammatory responses and
that DCs contribute more to Th1-type adaptive re-
sponses. The effective generation of Salmonella-specific
CD8+ T-cell responses and protection against subsequent
challenge in macrophage-depleted mice (46) support
the idea that APCs other than macrophages elicit
T-cell-mediated protection. Importantly, depletion of
macrophages from immune mice prior to challenge sig-
nificantly impairs protection as measured by bacterial
load in systemic organs (47). Thus, macrophages may
mediate protection primarily via bacterial clearance
rather than by APC function (36). It is also possible that
Ag presentation by macrophages may be more important
for stimulation of previously activated effector T cells
rather than naïve T cells (35).
Bacterial Interference with APC Function
As described above, adaptive immune responses to Sal-
monella infection require the function of professional
APCs. Surprisingly, Salmonella virulence depends on the
ability of this pathogen to survive and replicate within the
very cells required to mount immune responses against
it. This can be explained by the fact that coordinated
bacterial responses have evolved in the context of a host
immune system in which a generalized innate response to
bacteria leads to the development of a highly specific
adaptive response and memory. This coevolutionary
dynamic between pathogen and host has facilitated the
development of multiple mechanisms by which Salmo-
nella can subvert or evade APC function (references 48
and 49; also discussed below). The ability of Salmonella
to simultaneously survive within APCs and interfere with
the presentation of Ags to T cells contributes to virulence:
activation of adaptive immunity is inhibited while APCs
are exploited as protected reservoirs of replication and
means for bacterial dissemination.
In order for bacterial Ags to be acquired, processed, and
presented by APCs to T cells, they must be expressed by
bacteria in accessible cellular compartments and at bio-
logically significant levels. Together, these characteristics
have been described as Ag bioavailability (50). Bacterial
regulation of Ag bioavailability has been most extensively
studied for the dominant Ag FliC, a flagellar subunit
protein that is abundantly expressed on the bacterial
surface. Serovar Typhimurium actively regulates FliC
bioavailability during infection. The first level of this
regulation exists at the level of nongenetic variation; even
under conditions permissive for flagellar expression,
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serovar Typhimurium display a bistability phenotype in
which a significant number of bacteria in the population
are flagellin negative (51). Additional regulation of bio-
availability occurs during the transition from extracellu-
lar to intracellular environments within the host, as
serovar Typhimurium first restrict FliC expression to an
intracellular bacterial compartment (50) and then repress
FliC expression below the threshold required for T-cell
activation (52). Notably, Ag sequestration alone is not
sufficient to prevent processing and presentation of this
Ag by DCs; modifications of the bacterial surface are also
required (50). The global regulator PhoP, which is acti-
vated during intracellular growth (53) and required for
bacterial survival in the phagosome (54), is one mediator
of these modifications. PhoP regulates extensive re-
modeling of the bacterial surface which reduces its in-
flammatory properties, increases resistance to cationic
antimicrobial peptides, and alters membrane permeabil-
ity (55, 56, 57, 58). In the context of these modifications,
the processing and presentation of bacterial Ags re-
stricted to intracellular compartments is significantly
reduced (50). Importantly, repression of FliC expression
by intracellular-phase serovar Typhimurium is also PhoP
dependent (52, 59). Therefore, at a time when Salmonella
is residing and replicating within APCs, the organism
coordinately regulates the expression of genes essential
for both intracellular survival and the restriction of Ag
bioavailability to APCs.
In addition to alterations in Ag bioavailability, Salmo-
nella is able to directly interfere with APC function. For
example, Salmonella causes caspase-1-mediated pro-
inflammatory death (pyroptosis) of both macrophages
(16, 17) and DCs (18, 60) (see Chapter Nitric Oxide in
Salmonella and Escherichia coli Infections). Pyroptosis
represents one potential mechanism (destruction of
APCs) for avoiding the activation of adaptive immune
responses, but may also facilitate host responses by pro-
viding a source of antigenic material for bystander APCs
(61, 62). Salmonella also affects DC maturation: intra-
cellular-phase Salmonella inefficiently stimulates MHC-
II upregulation, costimulatory CD86 expression, and
secretion of proinflammatory cytokines such as TNF-α
and IL-12 (50). Virulent Salmonella may also inhibit Ag
processing and presentation on MHC-II by delaying
phagosome-lysosome fusion (63). This inhibition re-
quires expression of genes encoded in the Salmonella
Pathogenicity Island 2 (SPI2) and does not depend on Ag
uptake, MHC-II upregulation, or APC viability (40, 49).
The ability of Salmonella to interfere with APC function

may at least partially account for host restriction, because
only those serovars able to effectively prevent Ag pro-
cessing and presentation by DCs within a specific host
can limit activation of adaptive immune responses (64).
Importantly, it appears that Salmonella actively inhibits
APC function. SPI-2 mediated inhibition of Ag pro-
cessing and presentation requires live infection (40), and
live but not heat-killed PhoPc mutant bacteria (which
constitutively express PhoP in its activated state) inhibit
Ag processing by activated macrophages (45). Therefore,
at least some of the mechanisms that reduce Ag bio-
availability (PhoP-regulated surface modifications and
repressed Ag expression) also reduce the ability of APCs
to process and present Ag to T cells. Finally, Salmonella is
able to directly inhibit T-cell activation independent of
Ag processing and presentation (65), possibly by down-
regulation of TCR β-chain expression (66).
Summary: APCs and Salmonella-Specific Immunity
The processing and presentation of Ags to T cells is a
critical step in the development of adaptive immunity.
Importantly, Salmonella bacteria are not simply passive
collections of Ags, but rather actively modulate its in-
teractions with host cells. Multiple bacterium-directed
mechanisms, including altered Ag expression and bio-
availability, and interference with APC activation and
function, combine to modify Salmonella’s “pathogenic
signature” in order to minimize susceptibility to immune
surveillance (Fig. 1B, F, and H).
T CELLS
T Cells Are a Critical Component of Immunity to
Salmonella Infection
As a facultatively intracellular pathogen, Salmonella is
capable of replicating within host cells (10, 11), protected
from recognition by circulating antibodies. Therefore, a
robust T-cell response is required for both clearance of
primary infection and resistance to subsequent challenge
(67, 68, 69, 70). This section discusses the relative con-
tribution of various T-cell subsets to Salmonella-specific
immunity, the potential mechanisms by which they
mediate protection, and the identity of the Ags they
recognize.
T-Cell Subsets and Salmonella Immunity
T lymphocytes recognize Ag via heterodimeric receptors
(TCRs) expressed on their surface. The majority of T cells
circulating throughout the peripheral lymphoid system
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Adaptive Immune Responses during Salmonella Infection
express TCRs consisting of one α- and one β-chain. αβ T
cells can be further grouped according to the expression
of CD4 or CD8 coreceptors. CD4+ αβ T cells recognize
bacterial peptides from endocytic or phagocytic vacuoles
presented in the context of MHC-II molecules. They
function as “helper” T cells by producing cytokines that
stimulate B-cell antibody class switching, activation and
growth of cytotoxic T cells, and activation of phagocytes
such as macrophages (13). CD4+ T cells have been tra-
ditionally divided into Th1 and Th2 functional subsets
based on expression ratios of various cytokines that lead
to control of different classes of pathogens. However,
additional CD4+ T-cell subsets have recently been
identified (71, 72, 73, 74). CD8+ αβ T cells recognize
bacterial peptides from the cytosol presented by MHC-I
molecules. CD8+ T cells function as cytotoxic or “killer”
T cells that lyse infected host cells (13), and like CD4+ T
cells, are capable of secreting cytokines. In addition to αβ
T cells, a small percentage of T cells express a TCR
consisting of one γ- and one δ-chain and are concen-
trated in epithelial tissues, particularly in the gut mucosa
(75). γδ T cells express TCRs of limited diversity com-
pared with αβ T cells, do not appear to require MHC
presentation of protein Ags for their activation, and may
function in both innate and adaptive immune responses
(75).
Because infection of susceptible hosts with virulent Sal-
monella is rapidly fatal, most studies of the dynamics of
T-cell responses are performed with attenuated bacteria
(76). Salmonella-specific CD4+ and CD8+ T cells are
generated during infections with attenuated bacteria in
mice (77, 78, 79) and humans (80, 81, 82, 83, 84). T-cell
activation is quickly initiated after oral infection in mu-
cosal lymphoid tissues (85, 86). This is followed by the
expansion of a Salmonella-specific effector population in
the lymphoid tissues that is capable of redistribution to
nonlymphoid sites of infection (Fig. 2C) (77, 79, 80).
IFN-γ-producing CD4+ and CD8+ T cells can persist for
several months in mice and humans (43, 69, 82). In ad-
dition to αβ T cells, γδ T cells can also be detected after
Salmonella infection; a subset is activated at early stages
of infection via the MHC-Ib molecule Qa-1 (87, 88).
Many studies have confirmed that functional T cells are
required for the resolution of both primary and sec-
ondary Salmonella infections. For example, mice lacking
mature αβ T cells are severely impaired in their ability to
control primary oral infection with attenuated aroA
Salmonella (67, 88, 89, 70). Interestingly, T-cell function
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Cummings et al.
is not absolutely required in early stages of infection and
it appears that T-cell-independent mechanisms largely
mediate control of primary serovar Typhimurium in-
fection for up to 21 days (67, 90, 70). Immunized mice
that have been depleted of T cells are susceptible to
secondary virulent challenge despite antibody production
(67, 68, 69). Furthermore, transfer of immune T cells
(with serum) (7, 69) or T-cell lines derived from immune
mice (without serum) (91, 92) confers protection on
naïve susceptible hosts.
The pathogenesis of Salmonella infections is reflected in
the particular T-cell subsets most critical for host im-
munity. Because Salmonella replicates within phagocytic
vacuoles of infected cells it is not surprising that there is
an absolute requirement for CD4+ T cells in the control
of both primary and secondary Salmonella infection (67,
68, 69). The importance of functional CD4+ T cells is
highlighted by the increased frequency and severity of
nontyphoidal Salmonella infections correlating with re-
duced CD4+ T-cell counts in patients suffering from HIV
infection (93, 94, 95, 96, 97, 98). In addition, CD4+ T
cells also appear to actively suppress serovar Typhimu-
rium during chronic infection, as demonstrated by in-
creased recurrence of serovar Typhimurium bacteremia
in HIV patients (23, 99), or reactivation of infection in
CD4+ T-cell-depleted mice (100).
CD8+ T cells are generated during Salmonella infection of
mice and humans, and are restricted not only by MHC-Ia
molecules, but also nonclassical MHC-Ib molecules (81,
101, 102). There is evidence to suggest that Salmonella-
specific CD8+ T cells participate in protective immune
responses. During primary infection with attenuated
aroA Salmonella, MHC-I-deficient mice (lacking func-
tional CD8+ T cells) have higher bacterial loads during
primary oral infection than wild-type mice (87). Fur-
thermore, T-cell receptor knockout mice (no functional
CD4+ or CD8+ T cells) suffer from more severe infection
than CD4+ T-cell-deficient MHC-II knockouts (67),
suggesting that MHC-I-restricted CD8+ T cells partici-
pate in bacterial clearance. Additional studies indicate
that CD8+ T cells may actually be more important in
recall of immunity during secondary challenge. For ex-
ample, although naive β2m−/− mice (lacking functional
MHC-I molecules) are eventually able to resolve an initial
infection with attenuated bacteria, immune β2m−/− mice
are more susceptible than wild-type mice to virulent
challenge (78). Moreover, adoptive transfer of CD8+
T-cell-depleted immune cells provides limited protection
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to naïve mice compared with CD8+ replete control cells
(68, 69).
The contribution of γδ T cells to Salmonella immunity
remains unclear. In one study, γδ knockout mice were as
capable as wild-type mice of clearing infection with an
aroA-attenuated serovar Typhimurium strain (67); sim-
ilar results were observed after infection with Salmonella
enterica serovar Dublin (70). Other groups detected
slight defects in control of primary oral infection in γδ T-
cell-deficient mice, and increased numbers of γδ T cells
in the intestinal epithelium during infection (88). The
latter observation, together with the fact that γδ T cells
home to the gut mucosa (75), may indicate that γδ T cells
have some function during early stages of Salmonella
infection before systemic spread.
Mechanisms of T-Cell-Mediated Immunity
to Salmonella Infection
Although T cells are required for successful resolution of
Salmonella infections, the mechanisms whereby they
contribute to immunity are poorly understood. It is well
established that T cells generated in response to primary
Salmonella infection are capable of producing IFN-γ and
TNF-α (79). Studies using IFN-γ or TNF-α gene knockout
mice or antibody depletion of IFN-γ or TNF-α confirm
the importance of these cytokines in control and resolu-
tion of Salmonella infection (67, 68, 103, 104, 105, 106),
although the source of cytokines in these studies is not
clearly defined. These data support the idea that cytokine
production is the primary means by which T cells con-
tribute to immunity, and substantial evidence supports a
correlation between CD4+ T-cell function and IFN-γ
production. For example, CD4+ T-cell knockout mice,
which are unable to clear an infection with an aroA-at-
tenuated serovar Typhimurium strain, produce 100-fold
less IFN-γ than control mice (67). CD4+ T cells from mice
deficient in the IFN-γ transcription factor Tbet do not
produce IFN-γ or promote antibody isotype switching in
B cells; these mice are also susceptible to infection with
aroA-attenuated Salmonella (107). Interestingly, expo-
sure to lead induces a shift from a Th1 response to a Th2
T-cell response (with a corresponding shift from high to
low levels of IFN-γ and TNF-α production), thereby
rendering resistant C3H/HeN mice susceptible to oral
Salmonella infection (108). Others have demonstrated
IFN-γ production by gut-associated lymphoid tissue and
increased numbers of CD4+ and CD8+ T cells in the gut

during Salmonella infection (103, 109), suggesting that
localized expression of cytokines by T cells during pri-
mary infection may be important. Cytokine expression by
T cells may account for the ability of immune mice to
control a secondary infection with virulent Salmonella:
rapid release of cytokines by T cells primed to preferen-
tially home to the gut mucosa (110) facilitates rapid
macrophage activation and recruitment to the site of
initial host encounter with the pathogen (111). This idea
is supported by studies in which administration of anti-
TNF-α or IFN-γ antibodies exacerbated secondary viru-
lent infection in previously immunized mice (68, 104).
Finally, depletion of either IFN-γ or CD4+ T cells results
in reactivation of latent Salmonella infection (20, 100),
suggesting that IFN-γ may mediate CD4+ T-cell control
of latent infections.
Despite this evidence for the importance of cytokine
production by Ag-specific T cells in Salmonella infection,
other studies suggest the existence of IFN-γ–independent
T-cell actions. For example, it appears that cytokine
production alone is insufficient to induce clearance in the
absence of T cells: T-cell-depleted mice are susceptible to
challenge with attenuated aroA Salmonella despite high
serum levels of IFN-γ and TNF-α (68). Similarly, IL-12-
null mice fail to control infection with attenuated serovar
Typhimurium, despite equivalent IFN-γ and TNF-α
production by T cells in mutant and wild-type mice.
T cells in these mice did not proliferate in response to Ag,
indicating the importance of IFN-γ-independent, pro-
liferation-dependent T-cell responses in the control of
Salmonella infection (112). Together with observations
that NK-T cells and neutrophils have been identified as a
significant source of IFN-γ production in response to
serovar Typhimurium infection (113), and that T-cell
function is required only in the later stages of primary
sublethal infection (69), this finding suggests that T cells
are not the primary source of IFN-γ, at least during early
stages of infection (106).
Although the exact role of cytokine production by T cells
remains to be defined, there are other possible mech-
anisms by which T cells could mediate immunity to
Salmonella infection. For example, CD4+ T-cell help is
required to establish and maintain a functional CD8+
memory response (Fig. 1J) (114, 115). Salmonella-
specific CD8+ T cells are capable of lysing infected host
cells both in vitro (78) and in vivo (116). Therefore,
T-cell-mediated lysis of infected host cells could disrupt
the protected intracellular niche to render intracellular
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Adaptive Immune Responses during Salmonella Infection
bacteria accessible to phagocytes. In addition, CTL
granules contain the protein granulysin that has been
shown to have direct antimicrobial activity against Sal-
monella and other pathogens (117). Finally, CD4+ T-cell
help is required for B-cell functions such as isotype
switching and affinity maturation (Fig. 1K). For example,
in athymic mice (lacking mature T cells), there is a dra-
matically reduced LPS-specific antibody response (89).
CD28-deficient mice (with impaired T-cell activation)
exhibit diminished production of IgG1, IgG2a, or IgG2b
antibodies, and only low levels of IgM and IgG3 anti-
bodies (118). It may be that a combination of all of these
functions is required for optimal immunity to Salmonella
infection (Fig. 1J, K, and L), and further work remains to
define the exact importance of each contribution.
Antigenic Specificities of
Salmonella-Specific T Cells
Despite the well-established role for T cells in Salmonella
immunity, surprisingly little is known about the identity
or relative importance of the specific Ags recognized.
Perhaps the most thoroughly characterized Ag recog-
nized by Salmonella-specific CD4+ T cells is the flagellar
subunit protein FliC (43). This protein is abundant on
the cell surface (a single bacterium can have up to 10
flagella, each composed of 30,000 or more flagellin
molecules [119]) and is therefore an obvious target for
host immune responses. Indeed, multiple epitopes have
been identified within the FliC protein (120, 121, 122),
and a significant fraction of Salmonella-specific T cells
generated by sublethal infection recognize FliC (50, 123).
Immunization with purified FliC protein is protective
(123, 124). Significantly, the monomeric form of this
protein is a ligand for TLR5 (125, 126) and presumably
the Nod-like receptor Ipaf (127), and FliC is therefore a
target of both innate and adaptive immune responses.
FliC regulation is complex (51, 128) and acts to limit
adaptive immune responses: FliC is repressed below the T-
cell activation threshold during systemic growth (52). The
global regulator PhoP mediates this repression, and it may
therefore seem surprising that PhoPc bacteria (in which
FliC expression is constitutively suppressed) are strongly
immunogenic and prime protective immunity. However,
given the large number of potential proteins encoded in the
Salmonella genome, and a host T-cell compartment with
the capacity of generating at least 1018 different receptors
(129), it is likely that T cells responding to other
9
Cummings et al.
specificities are primed during infection and capable of
mediating protection. Indeed, analysis of proliferative re-
sponses to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)-fractionated whole bacteria
reveals that CD4+ T cells recovered from Salmonella-
immune mice respond to multiple non-FliC specificities
(52, 130). Despite this evidence, only a few additional
proteins have been definitively established as immune
T-cell targets: the Salmonella invasion protein SipC is
recognized by CD4+ T cells (131), and peptides derived
from GroEL are recognized by CD8+ cytotoxic T cells
(132) induced after natural infection.
Although the exact identity of additional Ags remains to
be determined, it appears that, like FliC, other surface-
exposed proteins are good candidates for Salmonella-
specific T-cell recognition. For example, bacterial extracts
enriched for surface Ags can prime protective immunity,
presumably mediated by IFN-γ production from im-
mune splenocytes and the generation of IgG2a antibodies
(133). T-cell lines derived from protectively immunized
mice respond to fimbrial proteins (134, 135). Salmonella
infection also primes T cells responding to outer mem-
brane proteins such as OmpC, porins, and iron-regulated
outer membrane proteins (IROMPS); immunization with
these proteins is protective and associated with increased
numbers of CD4+ and CD8+ T cells (136, 137, 138, 139).
Finally, it has recently been shown that membrane
vesicles (MVs) derived from the bacterial surface contain
Ags recognized by Salmonella-immune T cells, and im-
munization with purified MVs primes a protective CD4+
T-cell response (59, 140).
T-Cell Contributions to Salmonella Immunity:
Future Directions
Our understanding of T-cell responses to Salmonella
infection has dramatically improved in recent years.
Nonetheless, many questions remain to be answered.
What is the exact mechanism by which immune T cells
mediate protection? What is the complete antigenic
profile of T cells generated by protective immunization?
Do T and B cells recognize the same Ags? When and
where are protective T-cell responses generated? There is
a complex relationship between Salmonella and its hosts:
immune recognition of microbial Ags selects for patho-
gens capable of exploiting, modulating, or evading that
recognition in order to colonize and infect their hosts.
Therefore, identification of additional Ags recognized by
CD4+ T cells responding to infection will continue to
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provide insights into host functions required to resist
Salmonella infection.
B CELLS
The Role of B Cells in Salmonellosis
Natural oral Salmonella infection progresses from pene-
tration of the gastrointestinal epithelium to systemic
dissemination, generating B-cell and T-cell immune re-
sponses that collectively promote protective immunity
against virulent infection in susceptible hosts. While the
cell-mediated and humoral arms of the adaptive immune
response possess unique individual functions, interaction
between B and T cells is also critical. Antibody isotype
switching depends on T-cell help, and B cells contribute
to the priming of T cells and generation of T-cell mem-
ory. Thus, B-cell functions, including but not limited to
antibody production, are critical in the generation of
protective immunity against Salmonella infection.
Studies in B-Cell-Deficient Mice
The importance of B cells in immunity to serovar
Typhimurium has been explored by using mice devoid of
B cells (Igh-6−/−, Igμ−/−) (141). Igμ−/− mice possess a ge-
netic defect that disrupts the gene encoding the constant
region of the IgM heavy chain (μ-chain), rendering them
B-cell-deficient due to arrest in the stage of pre-B-cell
maturation (141). After intravenous (i.v.) or intraperi-
toneal (i.p.) infection, Igμ−/− mice control and clear an
attenuated serovar Typhimurium aroA strain from the
liver and spleen (142, 143, 144, 145). However, unlike
wild-type mice, Igμ−/− mice experience sepsis during
primary infection and are not protected against subse-
quent oral challenge with virulent serovar Typhimurium
(143, 145). Transfer of immune serum from wild-type
mice to Igμ−/− mice confers immunity to virulent chal-
lenge, underscoring the importance of B cells in the
generation of a protective immune response (144). Al-
though B cells do not appear to contribute significantly to
bacterial control during primary infection with aroA-
attenuated Salmonella, Igμ−/− mice are highly susceptible
to primary infection with virulent serovar Typhimurium
compared with wild-type mice (145). These data dem-
onstrate that B cells, or the products of B cells, are
necessary for both the initial control of primary viru-
lent serovar Typhimurium, and the development of
long-lived resistance against oral challenge with virulent
Salmonella.
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The Role of Secretory Antibodies in Protection
against Serovar Typhimurium (See Also Chapter
Mucosal Immune Responses to Escherichia coli
and Salmonella Infections)
Secretory IgA (sIgA) is present in mucosal tissues and
has been proposed to influence the pathogenesis of
mucosal pathogens (146). Salmonella initiate infection
via penetration of the mucosal epithelium, where colo-
nization of Peyer's patches (PPs) is required for gener-
ation of sIgA (9). The role of Salmonella-specific sIgA,
however, appears to be minimal in protective immunity.
Salmonella-specific monoclonal sIgA reduces adherence
of serovar Typhimurium to cells in vitro and confers
protection to naïve mice against oral challenge when
secreted by an implanted hybridoma tumor (147, 148).
However, although the presence of a sIgA response does
increase the dose necessary for initial PP colonization,
mice lacking the ability to generate sIgA are able to
generate a protective immune response equal to wild-
type mice, suggesting that serovar Typhimurium-specific
sIgA are not required for protective immunity (149). To
reconcile these seemingly conflicting data, it is important
to consider that the concentration of sIgA produced by
the hybridoma in vivo is likely very high, and may sig-
nificantly exceed the levels of Salmonella-specific sIgA
generated during immunization with viable attenuated
bacteria.
Innate sIgA antibodies are produced in response to
commensal microbiota, and their generation does not
depend on serovar Typhimurium infection. These anti-
bodies are reactive against a wide range of nonspecific
Ags, some of which likely cross-react with serovar
Typhimurium Ags (150). Both conventional B2 B cells
and CD5+ B1 B cells secrete innate sIgA antibodies (151).
While B2 B cells require direct interaction with CD4+ T
cells for stimulation of antibody secretion, B1 B cells do
not. Secretion of innate antibodies by B1 B cells is thought
to depend on the stimulating effects of bystander cyto-
kines such as IL-5, IL-6, and IL-10 (152). Unlike Salmo-
nella-specific sIgA (149), innate sIgA is protective against
natural Salmonella infection (153). In mice that lack the
ability to secrete IgA into their mucosa because of a defect
in the polymeric immunoglobulin receptor (pIgR−/−
mice), sIgA destined for secretion is instead retained in
the serum. pIgR−/− mice are more susceptible to serovar
Typhimurium infection via the natural oral route,
resulting in a higher bacterial load and increased fecal
shedding of Salmonella compared with wild-type mice.
Fecal shedding of serovar Typhimurium by pIgR−/− mice
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Adaptive Immune Responses during Salmonella Infection
also increases the incidence of transmission to normally
resistant wild-type mice, suggesting that innate sIgA may
play a role in “coating” bacteria present in feces, resulting
in decreased transmissibility of the organism (153).
The Influence of Salmonella-Specific
Antibodies on Protection
Susceptibility of mice to Salmonella infection depends on
several known genetic loci (154, 155, 156), and likely on
other strain-specific genetic signatures yet to be deter-
mined. These unique host factors are as influential as
pathogen-specific virulence factors in determining the
outcome of host-pathogen interactions. The role that
antibodies play in Salmonella immunity has been inves-
tigated in many different mouse strains, leading to a di-
verse data set. Eisenstein et al. (157) nicely demonstrated
that, even though naturally resistant and naturally sus-
ceptible mice are equally able to generate antibodies, the
resulting protective immunity against Salmonella was not
equal. This study provides a helpful framework for in-
terpretation of data exploring the role of Salmonella-
specific antibodies in protective immunity. To examine
the effects of host background on the generation of a
protective response against serovar Typhimurium, four
Salmonella-derived immunogens were administered i.p.
to the inbred mouse strains C3H/HeJ, C3H/FeJ, and
C3H/HeNCrlBR. Although each of these strains is de-
rived from the C3H lineage, they vary in their natural
susceptibility to Salmonella infection: C3H/FeJ mice are
hypersusceptible, C3H/HeJ are susceptible, and C3H/
HeNCrlBR mice are resistant. Whereas none of the
vaccines protected C3H/HeJ mice, and only administra-
tion of acetone-killed Salmonella generated significant
protection in C3H/FeJ mice, all four immunogens con-
ferred significant protection on resistant C3H/HeNCrlBR
mice. Transfer of immune sera from immunized C3H/
HeNCrlBR donor mice to naïve C3H/HeNCrlBR re-
cipients conferred complete protection against intraper-
itoneal challenge. However, the same serum transfer to
naturally susceptible mouse strains C3H/HeJ and C3H/
FeJ was unable to provide full protection against i.p.
challenge. Thus, the ability of antibodies to provide
protective immunity is strong when the host is naturally
resistant to Salmonella infection, but naturally suscepti-
ble hosts appear to require additional immune factors to
gain full protective immunity (see “T-cell subsets and
Salmonella immunity,” above). Therefore, the role of
antibodies in resistance to serovar Typhimurium infec-
tion depends on host susceptibility status.
11
Cummings et al.
In susceptible mouse strains, B cells appear to be most
important for protection during virulent serovar Typhi-
murium challenge. In mice with a functional T-cell re-
sponse, B cells are dispensable during primary i.v.
infection with aroA-attenuated Salmonella but necessary
for protection against subsequent i.p. or i.v. virulent
challenge (144). Igμ−/− and wild-type mice are equally
capable of clearing primary infection with aroA-attenu-
ated serovar Typhimurium, and mice of either back-
ground eliminate secondary i.v. challenge with attenuated
bacteria. Wild-type mice with functional B and T cells
clear secondary infection with attenuated organisms
more rapidly than Igμ−/− mice, indicating that optimal
bacterial clearance requires both memory T cells and B-
cell functions (144). However, Igμ−/− mice are less able to
control virulent Salmonella i.p. challenge, and Igμ−/− mice
immunized with attenuated bacteria become as resistant
to virulent challenge as wild-type animals when wild-type
immune serum is administered prior to challenge (144).
Thus, in susceptible mouse strains with functional T-cell
responses, B cells are dispensable during primary and
secondary infection with aroA-attenuated Salmonella
strains, but resistance to virulent serovar Typhimurium
infection via the i.p. and i.v. routes depends on the pre-
sence of B cells and/or immune serum constituents.
Consistent with these findings, Mastroeni et al. (143)
demonstrated that protection of naturally susceptible
mice against virulent oral Salmonella infection requires
both antibodies and T-cell responses. Together with the
data showing the necessity of antibody in protection
against virulent organisms administered i.p. and i.v.,
these observations confirm the importance of antibodies
in immunity against virulent serovar Typhimurium in
naïve susceptible hosts. The mechanism by which anti-
bodies provide protection remains unknown.
Naturally resistant mouse strains show a more variable
but still important dependence upon antibody response
for Salmonella-specific immunity. Administration of
immune sera protects naïve Salmonella-resistant C3H/
HeNCrlBR mice but not Salmonella-susceptible C3H/
HeJ and C3H/FeJ mice from infection (157). Immune B
cells and antibodies confer protection on otherwise sus-
ceptible male xid mice (harboring functionally defective
B cells but functional T cells) when transferred from
immunized Salmonella-resistant female littermates (158).
Therefore, antibody production as well as other non-
antibody-dependent B-cell functions (see below) aids in
protection against serovar Typhimurium, even in natu-
rally resistant hosts.
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Salmonella-Specific Antibody Responses
It has long been recognized that antibody responses are
generated against the abundant Salmonella surface Ag
LPS. However, it has become clear that antibody re-
sponses to protein antigens also contribute to protection,
although these responses have not been fully character-
ized. The complete repertoire of antibody specificities has
not been defined in the murine response or in humans
infected with serovar Typhi, although similarities likely
exist (159). In addition, the contributions of antibodies
with particular specificities in the generation of protec-
tive immunity have not been established. We focus below
on antibody responses generated by infection with live
Salmonella.
LPS, exposed on the surface of gram-negative bacteria,
elicits a strong antibody response during serovar Typhi-
murium infection (159, 160, 161). The majority of the
B-cell response to LPS targets the serotype-specific O-
antigen polysaccharide chains on the outermost part of
the molecule (162). The O-antigen likely obscures the
more internal lipid A and core structures from antibody
access. Conserved O-antigen residues, found across Sal-
monella serotypes, are less immunogenic than serotype-
specific residues, which may account for the inability of
anti-LPS antibodies to protect against challenge with
different Salmonella serotypes (163).
By using two serotypes of Salmonella with unique O-
antigens, Hormaeche et al. (163) elegantly demonstrated
that protection against oral Salmonella challenge depends
on antibody responses directed toward protein Ags in
addition to those against LPS moieties. Mice that were
i.v. immunized with either viable serovar Typhimurium
(O-antigen type O4) or Salmonella enterica serovar
Enteritidis (O-antigen type O9) and orally challenged
with the immunizing strain were protected. Conversely,
mice challenged with the Salmonella serotype not used
for immunization (termed the “heterologous” strain)
were not protected. The inability of heterologous strains,
distinguished by O-antigen type, to confer immunity
against one another indicates that LPS-specific immune
responses are responsible for protection. These results
suggested that immunization with serovar Enteritidis O4
would be fully protective against challenge with serovar
Typhimurium O4, and vice versa. However, only partial
protection is observed when mice are challenged with
heterologous bacteria expressing the O-antigen type of
the immunizing strain. These data are consistent with the
presence of antibodies targeting non-LPS (protein) Ags,
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as well as the idea that non-B-cell responses, such as
those from CD4+ T cells, also contribute to protective
immunity. Importantly, generation of antibodies against
protein Ags requires interaction with CD4+ T cells (see
“Mechanisms of T-cell-mediated immunity to Salmo-
nella infection,” above), and dependence on protein- and
non-protein-specific antibodies for complete protection
against Salmonella has been observed by several groups
(164, 165, 166). Taken together, these data indicate that
the Salmonella-specific antibody response generated
during protective immunity depends on multiple Sal-
monella Ags, including both LPS and protein(s). The
relative contribution of these specificities to protection
remains to be determined.
Defining the specificity of antibody responses resulting
from human serovar Typhi infection is likely to be com-
plex, particularly in regions of the world afflicted with
endemic typhoid fever. Anti-LPS antibody titers are al-
ready elevated in “naïve” subjects prior to immunization
or infection, potentially obscuring the significance of anti-
LPS antibody titers (167), and antibodies directed against
LPS in humans appear to be minor compared with anti-
bodies recognizing protein Ags. An inhibition enzyme-
linked immunosorbent assay (ELISA) method, in which
immune serum is preincubated with either outer mem-
brane proteins or LPS, was used to assess the ability of
these Ags to inhibit antibody binding to solid-phase
bound Salmonella Ags. Greater than 60% inhibition of
antibody binding to Salmonella Ags occurred after incu-
bation with low concentrations of soluble outer mem-
brane proteins, whereas preincubation with the highest
concentrations of LPS inhibited binding by less than 40%
(167). These data suggest that human anti-Salmonella
antibodies may be directed primarily toward protein Ags.
As in human infections, B cells also generate antibody
responses against a group of largely unknown protein Ags
during natural oral infection of mice. Proposed identities
of several B-cell antigens, based only on electrophoretic
mobility in one-dimensional SDS-PAGE followed by
Western blot analysis with immune sera, include OmpA,
outer membrane porins, flagellar subunit protein FliC,
outer membrane lipoprotein Lpp, and a cellular heat
shock protein (159).
Antibody responses to the outer membrane protein
OmpA resulting from infection have been experimentally
confirmed, and the predominant response is directed
against the C-terminal third of the protein (168). Sur-
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Adaptive Immune Responses during Salmonella Infection
prisingly, this epitope is proposed to lie in the periplasm,
indicating that the B-cell response is directed primarily
against a region of the protein that is not surface exposed
(168). However, OmpA has two structurally distinct
membrane conformations (169). The major OmpA spe-
cies is characterized by an N-terminal β-barrel in the
outer membrane and an α-helix rich C terminus in the
periplasm, whereas formation of the minor conformation
likely results in surface exposure of parts of the C ter-
minus (169). The C-terminal epitope is more immuno-
dominant than the N terminus during infection (170),
suggesting that surface exposure of the C terminus
provides the opportunity for B-cell recognition and an-
tibody production. The generation of anti-OmpA anti-
bodies also occurs in humans following serovar Typhi
infection, although the dominant epitope(s) have not
been determined (159, 167).
Infection has also been confirmed to prime antibody re-
sponses to outer membrane porins OmpC and OmpF
(165), and the dominant OmpC epitope is located on a
surface-exposed loop (162). During natural serovar Typhi
infection of humans, antiporin antibody titers increase
1,000-fold over control sera from uninfected subjects
(171), a phenomenon that has been confirmed by ad-
ditional studies by using both ELISA and immuno-
blot methods (167, 172). Any contribution of antiporin
antibodies to protective immunity, however, remains
unknown.
In addition to membrane proteins, abundant surface
appendages such as flagella and fimbriae are also targets of
the B-cell response. As an abundant extracellular Ag, Sal-
monella FliC elicits a strong antibody response in mice and
humans, in addition to its role as an important T-cell Ag
(see “Antigenic specificities of Salmonella-specific T cells,”
above) (173, 174). The murine antibody response to FliC is
generated following both oral and i.p. infection (173, 174).
Flagellin also induces antibody production in humans in-
fected with serovar Typhi (159, 171, 175). The antibody
response to fimbrial Ags depends on which fimbrial sub-
unit proteins are expressed during infection (176). Al-
though serovar Typhimurium grown in laboratory culture
prior to infection expresses only the FimA subunit, ex-
pression of ten different subunits occurs during murine
infection (176). Expression of various combinations of
these ten subunits occurs in vivo, with a corresponding
antibody response to the expressed subunit(s), but ex-
pression of all ten in an individual mouse is not observed.
These findings reinforce the concept that bacterial regu-
13
Cummings et al.
lation of Ag expression influences adaptive immune re-
sponses (52), and that the most relevant bacterial Ags to
investigate will not necessarily be obvious from the ex-
amination of bacteria grown in the laboratory setting.
Human B cells also generate an antibody response
against the Vi capsular Ag, which is expressed by serovar
Typhi and Salmonella enterica serovar Paratyphi C but
not serovar Typhimurium or serovar Paratyphi A and B.
Interestingly, whereas only 20% of patients with acute
typhoid fever have a detectable anti-Vi antibody titer,
approximately 90% of chronic gall bladder carriers
demonstrate an antibody response to the Vi Ag (reviewed
in reference 177). The dependence of temporal expres-
sion of Vi Ag in vivo on environmental signals suggests
that, like FliC, altered Vi Ag expression may affect
adaptive immune responses (see “Antigenic specificities
of Salmonella-specific T cells,” above). Immunization
with the Vi capsular Ag or live attenuated serovar Typhi
Ty21a provides protection against typhoid fever caused
by serovar Typhi (reviewed in reference 178). However,
these licensed typhoid fever vaccines do not provide
protection against serovar Paratyphi A infections (179,
180), and the coincident increase in incidence of
Vi-negative serovar Paratyphi A outbreaks in endemic
typhoid fever regions is consistent with the immuno-
dominance of the Vi Ag (180, 181, 182).
Identification of Salmonella B-cell Ags has proven diffi-
cult. Ags that have been most frequently characterized
are those expressed by bacteria grown in laboratory
culture conditions, which may not accurately represent
Ag expression and/or abundance in vivo. Selection of
bacterial targets that approximate what may be seen by
the host during infection is therefore a critical factor in
Ag discovery. Investigating outer membrane proteins
and surface appendages such as flagella and fimbriae has
yielded information about B-cell Ags exposed on the
bacterial cell surface. It is likely that important B-cell Ags
will be revealed with further investigation of other surface
organelles such as MVs (59), which are rich in outer
membrane and periplasmic proteins and elicit protective
responses when administered as an immunogen to both
mice and humans (140, 183).
B-Cell Functions in Addition to Antibody Production
B cells contribute to the generation of protective immu-
nity against serovar Typhimurium independent of anti-
body production. For example, B cells influence the
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immune response via secretion of cytokines (12, 184, 185,
186). Unlike splenocytes from immunized wild-type
mice, splenocytes harvested from immune B-cell-defi-
cient mice are unable to generate Th1-type cytokines
(IFN-γ, IL-2) in response to soluble serovar Typhimu-
rium Ags in vitro, despite the fact that T-cell populations
in both murine backgrounds are capable of cytokine
production (143). In the absence of B cells, the balance of
the adaptive immune response shifts away from the
characteristic Th1-type cytokine IFN-γ and toward the
production of the Th2-type cytokine IL-4 (27). B-cell
secretion of IFN-γ depends on combined stimulation of
TLR2, 4, and 9 (184). These observations suggest that B
cells support the generation of a Th1-type cytokine re-
sponse, either directly through cytokine production and
as APCs (see below) or indirectly through undefined
mechanisms, thereby facilitating the downstream am-
plification of the immune responses necessary for pro-
tection against virulent serovar Typhimurium.
B cells also contribute to Ag presentation in vivo (187,
188, 189, 190). Early in infection, DCs prime CD4+ T
cells through presentation of immunodominant peptides
in the context of MHC-II molecules (see “DCs and
macrophages as professional APCs during Salmonella
infection,” above). However, once CD4+ T cells are
primed, they are able to activate Ag-specific B cells, which
internalize Ag 10,000-fold more efficiently than non-
specific cells such as DCs do (191). Activated B cells may,
in turn, prime naïve T cells through presentation of a
much wider range of peptides from the original protein
Ag (189). Specifically, the ability of B cells to ingest,
process, and present Salmonella Ags depends on the
immune status of the host. For example, B cells harvested
from naïve mice or mice immunized 2 weeks prior to
harvest are refractory to infection in vitro, but B cells
isolated from mice immunized 3 months prior to harvest
can be infected by live serovar Typhimurium (27). B-cell
populations exhibit upregulation of the costimulatory
molecule CD86 and are able to present soluble serovar
Typhimurium Ags, whether they are obtained from na-
ïve, 2-week immune, or 3-month immune mice (192).
Such Ags are available for capture independent of the
bacteria from which they are derived, and may include
surface organelles such as MVs released by Salmonella in
vivo (193, 194). However, only B cells from 3-month
immune mice are able to present Ags derived from intact
heat-killed bacteria (27). Additionally, splenocytes har-
vested from wild-type mice are better able to present
Salmonella Ags to Salmonella-specific CD4+ T-cell lines
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than splenocytes of Igμ−/− mice or B-cell-depleted wild-
type splenocytes, demonstrating that B cells are an im-
portant APC population in the spleen (27). These data
suggest that B cells have unique Ag presentation capa-
bilities at different times postinfection, supporting a
model in which presentation of Ags to CD4+ T cells is
shared between DCs and B cells.
B-Cell Contributions to Salmonella Immunity:
Future Directions
It is clear that the generation of protective immunity
depends on multiple B-cell functions, including antibody
production, cytokine secretion, and/or Ag presentation
(Fig. 1K). B-cell involvement in protection against pri-
mary serovar Typhimurium infection and the generation
of protective immunity to secondary challenge may be
manifest at many stages of pathogenesis: during initial
migration to the intestinal epithelia, transition from the
intestinal lumen to the PP, uptake by other immune cells
such as macrophages, and/or progression to systemic
organs. The exact stage(s) during which B cells and/or
their products act, and how interactions occur between B
cells and Salmonella, are largely unknown. In addition,
the identity of Salmonella Ags that drive these responses
will be a key area of interest in future studies, with the
goal of determining the specificity of antibodies that in-
duce protective immunity.
TEMPORAL AND ANATOMICAL ASPECTS OF
SALMONELLA-SPECIFIC ADAPTIVE IMMUNITY:
FliC AS A MODEL Ag
During natural infection, Salmonella must survive the
intestinal environment (Fig. 2A), penetrate the mucosa of
the small intestine (Fig. 2B), colonize the PP (Fig. 2C),
and disseminate to systemic sites such as the liver and
spleen to replicate within host cells (Fig. 2E). The gut
mucosa presents a major initial barrier to Salmonella
dissemination after oral infection. In addition to over-
coming physical barriers such as the glycocalyx and
mucus layers, Salmonella must withstand chemical
attacks from antimicrobial peptides secreted by the in-
testinal mucosa (195, 196). Most Salmonella are con-
tained by innate host defenses in the small intestine or are
eliminated and do not progress farther than the intestinal
lumen. Although high doses of Salmonella (many times
the oral LD50) have often been employed in experimental
models of oral infection, only a few bacteria successfully
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Adaptive Immune Responses during Salmonella Infection
enter the systemic circulation when doses near the oral
LD50 are used (197, 198).
Flagella (and the flagellar subunit FliC) are abundantly
expressed on the surface of extracellular-phase Salmo-
nella (Fig. 2A) and therefore serve as a large reservoir of
ligands for both innate (125, 126) and adaptive (59, 43,
123) immune systems. After bacterial invasion or trans-
location of flagellin across the intestinal epithelial cell
layer, TLR5 stimulation results in a strong proinflam-
matory response that is not induced by normal
microbiota (Fig. 2B) (125, 199, 200, 201). Flagellin-
stimulated secretion of chemokines such as IL-8 (125,
199, 202) and CCL20 (203) results in the recruitment of
phagocytes, including DCs, to the small intestine (re-
viewed in reference 204). DCs are highly competent
APCs that can directly sample the gut lumen for Ags to
present to immune effector cells (8). In addition, flagellin
stimulates the maturation of both DCs and macrophages
(205, 206). Thus, the proinflammatory response to fla-
gellin, in addition to the proinflammatory response
evoked by Salmonella-induced cell death (207), results in
intestinal localization and activation of cells capable of
priming adaptive immune responses. APCs in the PP
(Fig. 2C) therefore initially prime CD4+ T cells that re-
spond to Ags expressed by bacteria invading the host
intestinal epithelium or residing in the gut lumen.
Small numbers of bacteria escape the gut and disseminate
by intracellular transport (9) to systemic sites such as the
liver and spleen (197, 198). There, Salmonella replicate
within macrophage phagosomes (10, 11), an intracellular
niche providing a safe haven from host antibodies. Sal-
monella have evolved complex regulatory systems to
precisely tailor gene expression to environmental con-
text. One global regulator of Salmonella gene expression
is PhoP, which is activated by intracellular growth con-
ditions and mediates virulence gene expression, bacterial
surface modifications, and repression of FliC, a dominant
Ag for Salmonella-specific CD4+ T cells (52, 53, 54, 55,
58).
Regulation of FliC expression is complex. During later
stages of Salmonella infection, when bacteria are repli-
cating intracellularly at systemic sites, FliC expression is
repressed below the T-cell activation threshold in a
PhoP-dependent manner (52). In addition to reduced
FliC expression, PhoP-mediated modifications of the
bacterial surface restrict the access of this Ag to APCs for
processing and presentation to T cells (50). The natural
15
Cummings et al.
consequence of this repression is that CD4+ T cells
primed to respond to Ags (such as FliC) that are ex-
pressed early in infection will fail to recognize intracel-
lular bacteria growing at systemic sites during later stages
of infection (Fig. 2E). Therefore, it appears that the co-
ordinated control of Ag expression and accessibility to
APCs (bioavailability) directly imposes temporal and
anatomical limits on the corresponding T-cell response.
This idea is supported by the findings that FliC-specific
CD4+ T-cell activation is restricted to the gut, despite
systemic bacterial colonization (86), and that bacteria
present in the PP but not MLN or spleen express FliC
protein. Importantly, Salmonella in the PP do not
homogenously express FliC; up to 40% of the bacteria are
FliC negative (51). Heterogeneous gene expression
provides a mechanism whereby one bacterial population
provides abundant Ags for T-cell priming, while a sub-
population already in “stealth mode” is poised to evade
the developing T-cell responses (Fig. 2C left).
Repression of protective Ags like FliC, in concert with
other genetically co-coordinated modifications of the
bacterial surface as described above, could facilitate
bacterial growth in vivo by affording Salmonella the
opportunity for additional rounds of replication before
the onset of effective adaptive immune recognition. This
is a reasonable explanation for the different outcomes
resulting from infection of naïve, susceptible mice with
virulent versus attenuated bacteria. Attenuated aroA-
mutant Salmonella grows more slowly in vitro and in
vivo (208, 209) and is cleared by immunocompetent
hosts, while virulent Salmonella rapidly overwhelms host
defenses. The growth advantage of virulent bacteria is
eliminated by prior immunization, as rapid activation of
localized host T- (and B-) cell functions provides pro-
tection (Fig. 1 right) (210, 211). In this context, it is
important to note that T cells from immune mice can
detect intracellular (and therefore FliC) bacteria (52).
However, it is not known when or where T cells
responding to Ags expressed by intracellular phase (FliC)
bacteria are primed, or whether these T cells are capable
of mediating protection. Interestingly, it has been shown
that systemic (spleen and liver) macrophages, although
capable of processing and presenting Ags to T cells, do
not affect the development of an adaptive T-cell response
(46). This suggests that APCs at systemic sites (Fig. 2E)
may not be capable of priming a T-cell response of suf-
ficient speed or magnitude to protect a host from sub-
sequent challenge with virulent Salmonella. From a host
perspective, a T-cell response localized to the gut and
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directed toward Ags expressed by extracellular bacteria at
early stages of infection (when the risk of systemic dis-
semination is greatest) appears to be an effective strategy.
This idea is supported by the high rate of nontyphoidal
Salmonella bacteremia in AIDS patients compared with
immunocompetent hosts (95, 96, 97). Studies using a
model peptide Ag indicate that systemic Salmonella-
specific CD8+ memory responses appear to be restricted
to Ags encountered in the gut mucosa (136). CD4+ and
CD8+ T-cell levels in the gut increase threefold during
secondary challenge of protectively immunized mice
(103). This is likely because DCs in the PP (the most
likely site of T-cell priming during primary infection) are
specifically able to imprint gut-homing specificity on T
cells (Fig. 2C) (110). Indeed, after oral immunization in
humans with serovar Typhi, most of the effector (IFN-
γ-producing) CD4+ and CD8+ T cells express the α4β7
gut-homing receptor (80). Finally, memory T cells ex-
press CCR6, the only known receptor for CCL20, a
chemokine secreted by intestinal epithelial cells after
stimulation by flagellin (212). Therefore, in an immu-
nized host, it appears that memory T cells primed to
respond to early-phase Ags expressed by extracellular
bacteria are recruited to the intestine where their
resulting activation could facilitate the containment of
bacterial replication and dissemination. In addition, it
seems likely that opsonic Ig would beneficially augment
the host response by facilitating Ag uptake, processing,
and presentation (Fig. 1P).
CONCLUDING REMARKS
As a facultatively intracellular pathogen, Salmonella is an
effective model organism for the study of adaptive im-
mune responses. Antibodies, the products of B cells,
opsonize extracellular bacteria for phagocytic uptake
(Fig. 1P), and Ig on the B-cell surface mediates high-
efficiency Ag capture for Ag presentation (Fig. 1K).
CD4+ and CD8+ T cells generated in response to Sal-
monella infection detect intracellular bacteria and are
required for bacterial clearance. Accordingly, full im-
munity to Salmonella infection in naïve, susceptible hosts
requires both T- and B-cell functions.
The requirement of both sera and T cells for immunity
suggests that, in addition to their individual functions,
the interaction between T and B cells is important. Al-
though there has traditionally been a differentiation be-
tween “cellular” and “humoral” immunity, in reality
these responses are tightly interconnected. T cells are
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required to activate B-cell isotype class switching and
affinity maturation. B cells may present Ags to T cells and
produce cytokines that support the development of a
Th1-type T-cell response (Fig. 1K). CD4+ T cells are
required to initiate activation of cytotoxic CD8+ T cells
(Fig. 1J). In addition, cytokines released by T cells may
activate APCs to more efficiently process and present
Ags, while cytokines released by activated APCs facilitate
T-cell activation and proliferation (Fig. 1E and L).
The interaction between Salmonella and its host is
complex and dynamic. The host mounts an immune
defense against the pathogen, which in turn acts to re-
duce, evade, or exploit these responses to successfully
colonize the host. Correspondingly, a greater under-
standing of adaptive immune responses provides tools
and reagents for gaining additional insights into mech-
anisms of bacterial pathogenesis. Salmonella bacteria are
not just passive collections of Ags, but rather actively
participate in their encounters with host cells. Multiple
bacteria-directed mechanisms, including altered Ag ex-
pression and bioavailability, interference with APC acti-
vation and function, and direct inhibition of T-cell
activation (65), combine to modify Salmonella’s “patho-
genic signature” to minimize susceptibility to immune
surveillance (Fig. 1B, F, and H).
Although our understanding of Salmonella-specific im-
munity has dramatically improved in recent years, many
unanswered questions remain. For example, the details of
intestinal DC function, T-cell priming, T-cell migration,
and the generation of T-cell memory in human serovar
Typhi infection remain incompletely understood. The
exact mechanisms by which T and B cells act to clear
Salmonella are unknown. Surprisingly little is known
about the identity, regulation, and relative contributions
to immunity of the individual bacterial Ags recognized by
Salmonella-specific B and T cells (the antigenisome). As
discussed above, emerging data suggest that bacterial
regulation of Ag expression and localization is critically
important: temporal and anatomical restrictions imposed
by bacterial regulation of Ag expression affect the de-
velopment of FliC-specific T-cell responses (50, 52). MVs
have recently been identified as bacterial organelles ca-
pable of priming protective immune responses in the
absence of infection (140). As nonreplicating structures
derived from the surface of genetically malleable orga-
nisms such as Salmonella, MVs could provide a powerful
new tool for both the analysis and delivery of immune
targets. Collectively, these data suggest that continued
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Adaptive Immune Responses during Salmonella Infection
identification of adaptive immune targets will guide
rational vaccine development, provide insights into the
host functions required to resist Salmonella infection,
and correspondingly provide valuable reagents for de-
fining the critical pathogenic capabilities of Salmonella
that contribute to its ability to cause acute and chronic
infections.
ACKNOWLEDGMENTS
No potential conflicts of interest relevant to this review were reported.
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