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Infection and Immunity, June 2007, p. 2708-2716, Vol. 75, No. 6
0019-9567/07/$08.00+0     doi:10.1128/IAI.01905-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Systemic CD8 T-Cell Memory Response to a Salmonella Pathogenicity Island 2 Effector Is Restricted to Salmonella enterica Encountered in the Gastrointestinal Mucosa

Departments of Microbiology,¹ Medicine, University of Colorado Health Sciences Center at Fitzsimons, Aurora, Colorado 80010,² Integrated Department of Immunology, National Jewish Research and Medical Center, Denver, Colorado 80206³

Received 1 December 2006/ Returned for modification 27 February 2007/ Accepted 20 March 2007

	ABSTRACT

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To better understand the evolution of a systemic memory response to a mucosal pathogen, we monitored antigen-specific OT1 CD8 T-cell responses to a fusion of the SspH2 protein and the peptide SIINFEKL stably expressed from the chromosome of Salmonella enterica and loaded into the class I pathway of antigen presentation of professional phagocytes through the Salmonella pathogenicity island 2 type III secretion system (TTSS). This strategy has revealed that effector memory CD8 T cells with low levels of CD62L expression (CD62L^low) are maintained in systemic sites months after vaccination in response to low-grade infections with Salmonella. However, the CD8 T-cell pool eventually declines. Low numbers of central memory cells surviving after prolonged resting from an antigen encounter can nevertheless reconstitute the systemic effector memory pool in a route-specific recall response to cognate antigens encountered in the gut. Accordingly, populations of CD62L^high interleukin-7 receptor-positive progenitor central memory cells grafted into naïve mice expand in response to orally administered Salmonella expressing the chromosomal translational fusion of sspH2 and the sequence encoding the SIINFEKL peptide but fail to proliferate following systemic stimulation. Moreover, populations of systemic memory CD8 T cells restricted to Salmonella in oral vaccines selectively expand in response to cognate antigens presented by cells isolated from mesenteric lymph nodes (MLN). Together, these findings have revealed the imprinting of systemic CD8 central memory T-cell recall responses against enteropathogens by MLN. MLN restriction represents a novel mechanism by which systemic CD8 T-cell immunity is confined to periods of high risk for extraintestinal dissemination.

	INTRODUCTION

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The gastrointestinal mucosa hosts the largest and most varied population of indigenous microbiota in the body and serves as a residence for a plethora of transient microorganisms and frank enteropathogens (18, 48). Some enteric pathogens such as Shigella species do not usually progress beyond the intestinal mucosa, whereas others, such as Salmonella and Yersinia species, not only invade the mucosal surface but can also be recovered from extraintestinal sites. The systemic dissemination of Salmonella enterica in humans is frequently associated with serovars Typhi and Paratyphi, which account for 22 million cases of bacteremia and 220,000 deaths a year worldwide (7). The extraintestinal dissemination of Salmonella, however, is not limited to typhoidal strains, as demonstrated by the fact that nontyphoidal salmonellae are the most common cause of hospital admission due to bacteremia in sub-Saharan Africa (16). Therefore, successful immunization against this class of disseminating enteropathogens must evoke long-lasting immunity in both local and systemic compartments of the immune system. Here we have studied long-term systemic memory responses to an oral vaccine containing Salmonella with early tropism for ileal phagocytes by using CD8 T-cell responses to an effector protein delivered into the host cell cytosol as an indicator of immunity.

CD8 T cells are an important component of the multiple cellular lineages required for optimal protective immunity against the intracellular pathogen Salmonella (13, 19, 25, 29). The differentiation of CD8 T cells has proven to be an excellent indicator of memory. Memory CD8 T cells expressing high levels of CD44 (the CD44^high phenotype) have been categorized into effector and central populations according to their patterns of L-selectin CD62L expression (38). In contrast to effector memory cells, long-lived central memory T cells capable of antigen-independent or homeostatic proliferation express high levels of the CD62L receptor. The patterns of CD62L expression by CD8 T cells have shown, for instance, that acute systemic Salmonella infections induce a protracted effector memory response (20). To study the type of systemic immunity induced by pathogenic microorganisms encountered in the gastrointestinal tract, we measured the CD8 T-cell memory response elicited by invasion-deficient Salmonella strains capable of extraintestinal dissemination (34, 44, 45). An oral vaccine containing an invasion-deficient Salmonella strain was engineered to inject the SIINFEKL model peptide into the cytosol of professional phagocytes as a chimera with the Salmonella pathogenicity island 2 (SPI2) SspH2 effector that is selectively expressed within endosomes. Systemic OT1 CD8 T cells specific to the SspH2::SIINFEKL chimera stably expressed from the Salmonella chromosome were used to monitor the progression of long-term systemic CD8 memory responses to mucosal stimulation.

	MATERIALS AND METHODS

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Bacterial strains. S. enterica serovar Typhimurium strain ATCC 14028s was used as the background for the construction of isogenic vaccine strains. The sspH2::SIINFEKL chromosomal translational fusion was constructed by following the one-step mutation procedure originally described by Datsenko and Wanner (9). The forward primer contained 60 bp of DNA homologous to the sspH2 target gene, followed by a sequence encoding amino acids 257 to 264 of the ovalbumin peptide (OVA_257-264; SIINFEKL) and the upstream FLP recombination target site of the pKD13 plasmid. DNA sequencing verified the insertion of the sequence encoding the OVA_257-264 peptide SIINFEKL in frame between nucleotides encoding residues 453 and 455 of SspH2 (Fig. 1A). The sspH2::SIINFEKL allele was inserted into Salmonella strains CL1509 (aroA) (40) and AJB82 (aroA invA) (4) to yield strains AV2199 (aroA sspH2::SIINFEKL⁺) and AV2200 (aroA invA sspH2::SIINFEKL⁺). The sseB::aphT mutation that inactivates the SPI2 translocon was moved from strain HH102 (12) into strain AV2200, yielding strain AV2201 (aroA invA sseB sspH2::SIINFEKL⁺).

View larger version (39K): [in this window] [in a new window]   FIG. 1. A model for studying CD8 systemic responses to oral Salmonella vaccines. (A) Gel showing PCR fragments of wild-type sspH2 and the sspH2::SIINFEKL translational fusion. Below, the OVA252-267 peptide sequence is shown in bold, flanking SspH2 domains lie within the green boxes, and the scar left after the excision of pKD13 is underlined. (B) The SIINFEKL peptide (black rectangle) is injected into the cytosol of APC via the SPI2 TTSS as an SspH2 chimera. After digestion, the SIINFEKL peptide is transported by TAP-1 into the endoplasmic reticulum (ER) for binding with major histocompatibility complex class I molecules (red). OT1 CD8 ß T-cell responses to the SIINFEKL peptide serve as surrogate markers of Salmonella-specific immunity. (C) Ly5.1+ C57BL/6 mice were orally immunized with an invasion-deficient Salmonella vaccine strain (aroA invA sspH2::SIINFEKL+) 1 to 3 days after the grafting of naïve Ly5.2+ CD8+ OT1 cells. The expression of Ly5.2 by CD8+ splenocytes was measured by flow cytometry. The SPI2 dependency of cytosolic translocation was studied by expressing the chimera in sseB mutant Salmonella cells. (D and F) Levels of IFN- in the sera of orally immunized mice (D) and in the supernatants of splenocytes cultured with the SIINFEKL peptide or Salmonella parboiled antigens (F) were measured by ELISAs. Because Salmonella cells disseminating extraintestinally reach the spleen and liver in identical numbers (45), the bacterial burden was quantified by plating all tissue from livers macerated in a stomacher. (E) The role of the cytosolic pathway in the SspH2::SIINFEKL-dependent stimulation of Ly5.1+ OT1 cells was tested with Ly5.2+ TAP-1 knockout (KO) mice. All responses were measured 7 days after oral boosting. The data are representative of five to eight independent observations from at least two separate experiments.

Adoptive transfers of OT1 cells and oral immunization. About 2 x 10⁶ nylon wool column-purified T cells isolated as described previously (33) from among splenocytes of OT1 transgenic mice were inoculated intravenously into naïve or immunized C57BL/6 mice. Mice receiving OT1 cell grafts were immunized orally with approximately 5 x 10⁹ CFU of Salmonella strain AJB82, AV2200, or AV2201 and were given a booster dose 2 weeks later. Selected groups of naïve or immunized mice received grafts of memory cells isolated from immunized animals 40 to 110 days after oral immunization. Memory CD8 T cells were isolated from splenocytes by nylon wool column purification, and the cell population was further enriched with memory CD8 T cells by positive magnetic-bead sorting using biotinylated anti-CD8 monoclonal antibodies and magnetic bead-labeled streptavidin (Miltenyi Biotech, Auburn, CA). CD8 memory cells were adoptively transferred into naïve controls or mice immunized 40 to 50 days earlier. Selected groups of mice were orally immunized a few days after grafting, whereas the water of some orally immunized mice was treated during the last 3 weeks with nalidixic acid before memory cells were grafted.

Immunoassays. SIINFEKL peptide-specific CD8 splenic responses were analyzed by flow cytometry by dually labeling CD8 and Ly5.1 or Ly5.2 in a lymphocytic gate designed according to forward and side scattering. Memory cells, identified on the basis of CD44^high expression, were classified further as central or effector memory lymphocytes according to their high or low levels of CD62L expression, respectively (37). The production of gamma interferon (IFN-) by splenocytes cultured in RPMI medium supplemented with 10% fetal calf serum (BioWhittaker, Walkersville, MD), 2 mM L-glutamine, 15 mM HEPES, 1 mM Na pyruvate (Sigma-Aldrich, St. Louis, MO), 5 µM 2-mercaptoethanol, 100 U of penicillin/ml, and 100 mg of streptomycin (Cellgro, Herndon, VA)/ml in the presence or absence of 0.50 µg of the SIINFEKL peptide/ml was measured by sandwich enzyme-linked immunosorbent assays (ELISAs). All the reagents for flow cytometry and the ELISAs were purchased from Caltag (Burlingame, CA), BD Pharmingen (San Diego, CA), or eBiosciences (San Diego, CA).

Isolation of APC. Antigen-presenting cells (APC) were isolated from the spleens, Peyer's patches, or mesenteric lymph nodes (MLN) of C57BL/6 mice. Spleens were macerated and the red blood cells were eliminated by using Gey's lysis buffer. MLN and Peyer's patches were resected aseptically and digested in Dulbecco's phosphate-buffered saline, without Ca²⁺ or Mg²⁺ (Sigma-Aldrich), containing 0.2% collagenase I and IV (Sigma-Aldrich) and 2% heat-inactivated fetal calf serum for 5 min at 37°C in a 5% CO₂ incubator. The specimens were placed on ice for 10 min in 5 mM EDTA diluted in Dulbecco's phosphate-buffered saline supplemented with 2% heat-inactivated fetal calf serum. Cells from MLN were pelleted and resuspended in RPMI medium as described above for splenocytes.

Antigen presentation. Cells (4 x 10⁵) isolated from spleens, Peyer's patches, and MLN were added to 24-well plates containing 10⁶ OT1 cells. OT1 cells were isolated from mice immunized orally between 70 and 110 days earlier with an aroA invA sspH2::SIINFEKL⁺ Salmonella vaccine strain. OT1 cells contained in the splenocyte samples were nylon wool-purified, and the samples were further enriched with OT1 cells by positive selection for CD8 T cells by magnetic-bead cell sorting according to the indications of the bead manufacturer (Miltenyi Biotech). APC and OT1 cells were cultured for 3 days with 0.50 µg of the SIINFEKL peptide/ml at 37°C in a 5% CO₂ incubator. The percentages of CD8⁺ (fluorescence parameter FL1) and Ly5.1⁺ (fluorescence parameter FL3) OT1 T cells contained in a lymphocyte gate were determined by flow cytometry after Topro-3⁺ (fluorescence parameter FL4) dead cells were eliminated from the analysis.

CD8 T-cell proliferation. Selected groups of naïve or memory OT1 cells were labeled as previously described (21) with 5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) prior to the adoptive transfer. Levels of proliferating CD8⁺ Ly5.1⁺ T cells were estimated by using a four-color FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) to monitor the loss of CFSE 5 to 21 days after transfer. Dead cells were eliminated from the analysis after the gating out of Topro-3⁺ (FL4) cells.

	RESULTS

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Stimulation of antigen-specific CD8 systemic T-cell responses by an orally administered Salmonella vaccine strain expressing the sspH2::SIINFEKL translational fusion. The acquired response against Salmonella involves B cells and T cells as well as CD4 and CD8 ß T cells (13, 19, 23-25, 29, 30). Several investigators have successfully measured acute CD8 T-cell responses to Salmonella expressing OVA from a plasmid (11, 39, 49). However, in the absence of selective pressure, plasmids are frequently lost and thus are less reliable for long-term studies. To examine long-term systemic responses to oral Salmonella vaccines, we engineered a stable OVA_257-264 SIINFEKL construct expressed from the Salmonella chromosome as a chimera with an SPI2 effector that allows long-term expression in vivo (Fig. 1A). Because substrates of the type III secretion system (TTSS) are frequently poor immunogens, SPI2 TTSS effectors were evaluated for homology to the SPI1 SptP effector that is known to stimulate T-cell responses (35). Protein sequence alignments to determine homology to SptP domains identified the SspH2 effector, which is secreted into the host cell cytosol in an SPI2-dependent fashion (27, 28). The SIINFEKL peptide inserted in frame in a predicted flexible loop of SspH2 (Fig. 1B) was expressed in an aroA invA Salmonella vaccine strain (45).

Similar to CD8 T cells involved in the acute responses triggered by Salmonella expressing high levels of OVA (49), antigen-specific Ly5.2⁺ CD8⁺ T cells accumulated in the spleen shortly after oral immunization with the Salmonella vaccine strain expressing the sspH2::SIINFEKL chromosomal translational fusion (Fig. 1C). Further analysis verified that the Ly5.2⁺ CD8⁺ T cells expressed the Vß5.1 allele characteristic of OT1 transgenic cells (data not shown). To demonstrate dependency on the SPI2 TTSS for the delivery of the SspH2::SIINFEKL chimera into the cytosol, the sspH2::SIINFEKL allele was expressed in an isogenic aroA invA vaccine strain carrying the sseB::aphT mutation that inactivates all intracellular SPI2-mediated translocation. The sseB mutant strain triggered significant (P < 0.01) Salmonella-specific IFN- systemic responses, although it was less immunogenic than the corresponding SPI2-proficient control (Fig. 1D, left panel). In sharp contrast to its IFN--stimulating capability, the sseB mutant vaccine strain failed to trigger any significant (P = 0.32) expansion of the population of Ly5.2⁺ CD8⁺ cells (Fig. 1C). The lack of a CD8 T-cell response cannot be explained by reduced survival and extraintestinal dissemination of the sseB-deficient vaccine strain, since mice inoculated orally with either sseB-proficient or sseB-deficient vaccine strains harbored identical hepatic bacterial loads (P = 0.87) (Fig. 1D, right panel). The lack of SIINFEKL peptide-specific CD8 T-cell responses in mice immunized orally with the aroA invA sseB sspH2::SIINFEKL⁺ vaccine strain is consistent with the SPI2-dependent translocation of SspH2 into the cytosol (27, 28).

TAP-1-deficient mice were used to test whether CD8 T-cell responses elicited by the Salmonella vaccine require the translocation of the SIINFEKL peptide from the cytosol into the endoplasmic reticulum. Because TAP-1-deficient mice express the Ly5.2 allele, the OT1 transgene was backcrossed into an Ly5.1⁺ C57BL/6 background. Ly5.1⁺ OT1 transgenic mice were used for this and all subsequent experiments. Figure 1E shows that the TAP-1 transporter is critical for the expansion of populations of antigen-specific CD8⁺ T cells derived in response to the orally administered sspH2::SIINFEKL⁺ Salmonella vaccine strain. In agreement with the results of other studies (1, 41), an endogenous Ly5.1^– CD8⁺ population consisting mainly of T cells was nevertheless observed in the spleens of immunized TAP-1-deficient mice. In contrast, ß T cells represented most of the Ly5.1^– CD8⁺ lymphocytes in wild-type mice. Splenocytes isolated from orally vaccinated TAP-1-deficient mice did not secrete IFN- in response to the SIINFEKL peptide but synthesized amounts of IFN- similar to those synthesized by TAP-1-competent controls in response to parboiled Salmonella antigens (Fig. 1F). Together, these findings are consistent with a model in which the SIINFEKL peptide is injected into the cytosol via the SPI2 TTSS as a chimera with SspH2, translocates into the endoplasmic reticulum for loading into major histocompatibility complex class I molecules, and is highly immunogenic. This strategy was used to evaluate long-term systemic CD8 T-cell memory responses evoked by an oral Salmonella vaccine.

Oral immunization elicits systemic effector memory CD8 T-cell responses. The kinetics of systemic CD8 T-cell responses to oral immunization with Salmonella were studied. In contrast to the relatively slow responses seen when OVA is delivered into the phagosome by systemically administered wild-type Salmonella (20), a six- to sevenfold increase in antigen-specific CD8 T cells in mice receiving OT1 cell grafts was already detected 7 days after oral exposure to sspH2::SIINFEKL⁺ Salmonella (Fig. 2A). The percentage of SIINFEKL peptide-specific T cells increased 15-fold 2 weeks after oral immunization and had increased about 65-fold by 7 days after oral boosting. The increase in Ly5.1⁺ CD8⁺ T cells was antigen specific and not the product of the expansion of populations of bystander T cells, because populations of OT1 T cells did not expand in mice immunized orally with an isogenic vaccine strain (i.e., an aroA invA strain) expressing the sspH2 wild-type allele (Fig. 2A). To determine whether the expansion of populations of antigen-specific CD8 T cells is a consequence of T-cell proliferation, donor OT1 cells were labeled with CFSE before grafting. As shown in Fig. 2B, all the grafted cells proliferated 3 weeks after oral immunization. Over 90% of the cells that proliferated in the spleens exhibited the CD44^high memory phenotype, whereas 80% of them were CD62L^low (Fig. 2C and D). Predominantly CD62L^low expression by CD44⁺ cells suggests that oral Salmonella vaccines preferentially trigger early effector memory T-cell responses.

View larger version (48K): [in this window] [in a new window]   FIG. 2. Effector memory CD8+-T-cell responses to an oral Salmonella vaccine. (A) The percentage of CD8+ Ly5.1+ SIINFEKL peptide-specific T cells recovered from the spleens of mice receiving grafts of OT1 cells was determined at different time points after oral immunization with aroA invA sspH2::SIINFEKL+ Salmonella. Naïve mice and mice inoculated orally with aroA invA Salmonella were used as controls. 7d, 7 days. (B) The proliferation of lymphocytes in the CD8+ Ly5.1+ gate was visualized as the loss of CFSE among cells from naïve or orally immunized (immune) mice 21 days after immunization. (C and D) The abundance of central memory cells was expressed as the percentages of CD8+ Ly5.1+ splenocytes that were CD44high (C) and CD62Lhigh (D). Regions were drawn by using isotype controls (data not shown) according to the CD44 and CD62L expression patterns of naïve OT1 cells. The mice in these experiments were immunized orally with the aroA invA sspH2::SIINFEKL+ Salmonella vaccine strain. The data are representative of nine independent observations from three separate experiments.

View larger version (37K): [in this window] [in a new window]   FIG. 3. Effector memory responses to oral Salmonella vaccines. (A) The frequencies of CD8+ Ly5.1+ cells in naïve mice (N) and orally immunized mice 40 days postimmunization (I) and those in orally immunized mice after intraperitoneal boosting with the aroA invA sspH2::SIINFEKL+ Salmonella vaccine strain (I-B) were determined. (B and D) The percentages of CD62Lhigh central memory (CM) and CD62Llow effector memory (EM) cells (B) and the hepatic bacterial burdens (D) in animals 40 days postimmunization after intraperitoneal boosting with 100 CFU were determined. (C) Splenocytes from immunized mice were pulsed in vitro with the SIINFEKL peptide and analyzed for their IFN--secreting capacities. The data in all panels are representative of six independent observations from two separate experiments.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Memory CD8 T cells are capable of both homeostatic and antigen-driven proliferation. (A and B) Ly5.1+ splenocytes isolated 40 days after animals were orally immunized (mem; panel A) or Ly5.1+ cells isolated from naïve controls (naïve; panel B) were grafted into naïve mice or mice that had been orally immunized 45 days earlier (immune). Some immune animals were treated with nalidixic acid in drinking water 3 weeks before grafting (immune+ATB). The frequency of SIINFEKL peptide-specific Ly5.1+ CD8+ splenic T cells was estimated by flow cytometric analysis. (C) CSFE-labeled naïve splenocytes or Ly5.1+ splenocytes taken from immune mice 45 days postimmunization (mem) were grafted into either orally immunized mice 45 days postimmunization (imm) or naïve recipients. Selected groups of mice were orally inoculated with the sspH2::SIINFEKL+ Salmonella vaccine strain 2 days after grafting (imm+boost). Cell division was evaluated by flow cytometry as the loss of the CFSE label among the CD8+ Ly5.1+ population. All responses were measured 7 days after grafting. The data are representative of 5 to 10 independent observations from at least two separate experiments.

View larger version (15K): [in this window] [in a new window]   FIG. 5. The antigen-driven expansion of CD8 memory cells is restricted to Salmonella cells encountered in the oral mucosa. (A) The percentages of CD8+ Ly5.1+ splenocytes in mice receiving grafts of OT1 cells were determined 50 days (50d) and 110 days (110d) after oral vaccination. An analysis was also performed 7 days after mice received oral (110d+po) or parenteral (110d+ip) booster vaccines at 110 days postimmunization. (B) The IFN- production in splenocytes isolated 50 or 110 days after oral immunization was measured. The SIINFEKL peptide (SIIN) was added to selected groups of splenocytes. The right panel shows the IFN- response 7 days after animals received oral booster doses at 110 days postimmunization. (C) Cells isolated from high responders (immune) were analyzed by flow cytometry for intracellular IFN-. OT1 cells transferred into naïve mice were used as controls. (D) CD8+ Ly5.1+ cells isolated from mice with elevated or reduced numbers of OT1 cells (i.e., high responders [H.R.] and low responders [L.R.], respectively) were examined for the expression of the interleukin-7 receptor (IL7R) and CD62L 110 days postimmunization. The data are representative of four to seven independent observations from two independent experiments.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Central memory CD8 T cells restricted to orally administered antigens drive systemic effector memory responses. (A) Ly5.1+ cells isolated from mice orally immunized with Salmonella 70 to 110 days earlier were grafted into naïve mice after positive selection over a magnetic bead column. The expansion of the Ly5.1+ CD8+-T-cell population was tested 7 days after the intraperitoneal (ip; middle panel) or oral (po; right panel) administration of an aroA invA sspH2::SIINFEKL+ Salmonella vaccine strain. (B) CD62Llow and CD62Lhigh CD8+ Ly5.1+ cells isolated by flow cytometric sorting from mice orally immunized 110 days earlier with aroA invA sspH2::SIINFEKL+ Salmonella were grafted into naïve mice. A day after grafting, selected groups of mice were either intraperitoneally or orally immunized with aroA invA sspH2::SIINFEKL Salmonella. The abundance of CD62L CD8+ T cells in a Ly5.1+ gate was analyzed by flow cytometry 7 days after Salmonella challenge. The data are representative of five independent observations from two separate experiments.

View larger version (24K): [in this window] [in a new window]   FIG. 7. Selective expansion of central memory cells restricted to antigen administered orally in response to cells isolated from MLN. APC from Peyer's patches, MLN, or spleens were cocultured for 7 days with OT1 memory CD8 T cells isolated from spleens of mice immunized orally 85 to 90 days earlier. The T-cell population was enriched with OT1 cells by CD8 magnetic-bead sorting of nylon wool-purified T cells. The expansion of transgenic cells was quantified by flow cytometric analysis by monitoring the frequency of CD8+ Ly5.1+ cells. The data are representative of 12 independent observations from three separate experiments.

	DISCUSSION

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The selective expansion of populations of SspH2::SIINFEKL-specific CD8 T cells following oral stimulation indicates that the SspH2 SPI2 effector is expressed in cells populating the gut mucosa. In vitro cultures of gut APC have identified MLN as the anatomical location where the restriction of SspH2::SIINFEKL-specific memory CD8 T cells to orally encountered antigens takes place. MLN restriction of CD8⁺ memory responses to the SspH2::SIINFEKL chimera is analogous to but different from the selective imprinting for gut-homing T cells by Peyer's patches (32), since these SspH2::SIINFEKL-specific memory CD8 T cells do not appear to be restricted by Peyer's patches. Differences in the anatomical restriction of memory cells to selected lymphoid tissues of the intestinal mucosa exemplify the functional compartmentalization of the inductive sites of the gut. Imprinting by Peyer's patches may be critical for directing recall effector and memory responses to the gastrointestinal mucosa, whereas MLN restriction may orchestrate temporal responses in distal sites during times of intestinal infection.

It should be noted, however, that not all systemic responses elicited in response to oral immunization are necessarily limited to antigens encountered in intestinal lymphoid tissues. In fact, a population of CD8^– Ly5.1⁺ donor cells primed during oral vaccination expanded following systemic boosting. Moreover, polyclonal populations of splenocytes selected by oral immunization do indeed proliferate in response to Salmonella antigens presented by APC from spleens (24). The recently described delayed extraintestinal route of dissemination that proceeds without previous colonization of Peyer's patches and MLN (3) may evoke systemic responses that are not restricted by intestinal lymphoid organs. The segregation of APC in defined anatomical compartments resembles the distribution of populations of Salmonella-specific, polyclonal memory CD4⁺ and CD8⁺ T cells at distinct anatomical locations according to functional capabilities (17). Together, these data illustrate the complexity in the mucosal and systemic compartments of the immune system. Immunity to enteropathogens involves local and distal inductive sites, which together with distinct T-cell migration patterns provide temporal and spatial diversification in the immune response required to effectively defend against enteropathogens with tropism for both mucosal and systemic organs.

An effector memory response was maintained in a few animals over 110 days. Most of these effector cells expressed the interleukin-7 receptor, suggesting that at late time points effector cells are committed to becoming long-lived memory cells (15). The inhibition of CD8 population expansion after antibiotic therapy and the expansion of populations of naïve cells in hosts that were immunized orally 50 days earlier (Fig. 4) suggest that a focus of infection maintains the lingering effector response. MLN phagocytes, which have been shown to facilitate the prolonged survival of enteric commensals and wild-type Salmonella (22, 31), may keep Salmonella vaccine cells viable for extended periods of time. These observations consequently indicate that the duration of systemic CD8 memory responses elicited by enteropathogens depends on microbial persistence in the host. Efficacious live-pathogen vaccines must therefore balance virulence potential and the capacity of vectors to cause chronic infections with the development of long-lasting immunity.

Populations of systemic memory CD8 T cells, nonetheless, decline overtime. The loss of effector memory may explain the advised reimmunization of seasonal travelers visiting areas of endemicity as well as the progressive fading of immunity in residents that depart from such areas for extended periods of time. The smoldering Salmonella infection does not appear, however, to induce the partial exhaustion of antigen-specific lymphocytes, because memory cells are capable of homeostatic and antigen-driven proliferation. The persistence of T-cell function suggests that the level of antigenic stimulation associated with Salmonella must be significantly lower than that associated with pathogenic microorganisms linked to T-cell deletion (46). In addition, the IFN- response seen here and elsewhere (23, 25) may inhibit SPI2 transcription via nitric oxide (26), thus limiting the expression of the SspH2::SIINFEKL chimera at later times. The adjuvanticity of Salmonella's outer membrane may also trigger inflammatory signals that preserve T-cell function despite enduring antigenic stimulation.

Several properties associated with the SspH2::SIINFEKL chimera have made it possible to elucidate the anatomical restriction of long-term central memory CD8 T cells elicited by the oral Salmonella vaccines used in this study. First, the SIINFEKL epitope is transcribed from a single sspH2 locus in the Salmonella chromosome, so CD8 OT1 responses in this system are sensitive surrogate markers of Salmonella-specific immunity to an SPI2 TTSS effector. Second, the chromosomal location of this translational fusion guarantees high stability, allowing studies of long-term immunity in vivo. Third, the cytosolic secretion of SPI2 substrates such as SspH2 is selectively induced within the Salmonella-containing vacuole (5, 10). SPI2-based systems, therefore, provide significant advantages for studying systemic responses compared to delivery systems based on a flagellar TTSS that is downregulated extraintestinally (8). And fourth, translation-secretion coupling of TTSS effectors such as SspH2 ensures the exclusive translocation of the chimera into the cytosol (2, 6), minimizing both antigenic build-up in phagosomes and cross-presentation from endocytic vacuoles. The direct loading of an antigen into the cytosolic pathway of antigen presentation may initiate faster CD8 T-cell priming and recall responses than those elicted by the intraphagosomal delivery of OVA (20). The SspH2 delivery system described herein can be used for the detailed characterization of mucosal and systemic responses to Salmonella and can be easily adopted to deliver a variety of heterologous antigens to the inductive sites of both the mucosal and systemic compartments of the immune system.

In summary, our findings have exposed the previously unrecognized MLN restriction of central memory CD8 T cells to Salmonella encountered in the gastrointestinal tract. MLN restriction may limit rapid and focused systemic memory CD8 T-cell recall responses to periods during which enteropathogens populate their natural niches in the host. The robust contraction of populations of CD8 effector memory cells that occurs over time may be advantageous in the context of intestinal immunity. Effector cells may wane with the disappearance of luminal cognate antigens, freeing space and resources for rising clones with specificity for the latest pathogenic challenges encountered in the intestinal mucosa. Central memory CD8 T cells restricted to orally encountered antigens that survive the contraction phase are nonetheless long lived and selectively reconstitute a labile pool of effector memory cells in systemic compartments of the immune system in response to cognate pathogens reaching MLN.

	ACKNOWLEDGMENTS

 
Support of this work was provided by the National Institutes of Health (AI54959, AI053213, RR16082) and the Schweppe Foundation.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Microbiology, Mail Box 8333, UCHSC School of Medicine at Fitzsimons, P.O. Box 6511, Room P18-9120, Aurora, CO 80010. Phone: (303) 724-4218. Fax: (303) 724-4226. E-mail: andres.vazquez-torres{at}uchsc.edu

Published ahead of print on 2 April 2007.

Editor: A. D. O'Brien

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1.	Aldrich, C. J., H. G. Ljunggren, L. Van Kaer, P. G. Ashton-Rickardt, S. Tonegawa, and J. Forman. 1994. Positive selection of self- and alloreactive CD8+ T cells in Tap-1 mutant mice. Proc. Natl. Acad. Sci. USA 91:6525-6528.[Abstract/Free Full Text]
2.	Anderson, D. M., and O. Schneewind. 1997. An mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science 278:1140-1143.[Abstract/Free Full Text]
3.	Barnes, P. D., M. A. Bergman, J. Mecsas, and R. R. Isberg. 2006. Yersinia pseudotuberculosis disseminates directly from a replicating bacterial pool in the intestine. J. Exp. Med. 203:1591-1601.[Abstract/Free Full Text]
4.	Baumler, A. J., R. M. Tsolis, P. J. Valentine, T. A. Ficht, and F. Heffron. 1997. Synergistic effect of mutations in invA and lpfC on the ability of Salmonella typhimurium to cause murine typhoid. Infect. Immun. 65:2254-2259.[Abstract]
5.	Chakravortty, D., M. Rohde, L. Jager, J. Deiwick, and M. Hensel. 2005. Formation of a novel surface structure encoded by Salmonella pathogenicity island 2. EMBO J. 24:2043-2052.[CrossRef][Medline]
6.	Chilcott, G. S., and K. T. Hughes. 2000. Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar Typhimurium and Escherichia coli. Microbiol. Mol. Biol. Rev. 64:694-708.[Abstract/Free Full Text]
7.	Crump, J. A., S. P. Luby, and E. D. Mintz. 2004. The global burden of typhoid fever. Bull. W. H. O. 82:346-353.
8.	Cummings, L. A., W. D. Wilkerson, T. Bergsbaken, and B. T. Cookson. 2006. In vivo, fliC expression by Salmonella enterica serovar Typhimurium is heterogeneous, regulated by ClpX, and anatomically restricted. Mol. Microbiol. 61:795-809.[CrossRef][Medline]
9.	Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.[Abstract/Free Full Text]
10.	Deiwick, J., T. Nikolaus, S. Erdogan, and M. Hensel. 1999. Environmental regulation of Salmonella pathogenicity island 2 gene expression. Mol. Microbiol. 31:1759-1773.[CrossRef][Medline]
11.	Harding, C. V., and J. D. Pfeifer. 1994. Antigen expressed by Salmonella typhimurium is processed for class I major histocompatibility complex presentation by macrophages but not infected epithelial cells. Immunology 83:670-674.[Medline]
12.	Hensel, M., J. E. Shea, S. R. Waterman, R. Mundy, T. Nikolaus, G. Banks, A. Vazquez-Torres, C. Gleeson, F. C. Fang, and D. W. Holden. 1998. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages. Mol. Microbiol. 30:163-174.[CrossRef][Medline]
13.	Hess, J., C. Ladel, D. Miko, and S. H. Kaufmann. 1996. Salmonella typhimurium aroA– infection in gene-targeted immunodeficient mice: major role of CD4+ TCR-alpha beta cells and IFN in bacterial clearance independent of intracellular location. J. Immunol. 156:3321-3326.[Abstract]
14.	Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, and F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76:17-27.[CrossRef][Medline]
15.	Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, and R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4:1191-1198.[CrossRef][Medline]
16.	Kankwatira, A. M., G. A. Mwafulirwa, and M. A. Gordon. 2004. Non-typhoidal salmonella bacteraemia—an under-recognized feature of AIDS in African adults. Trop. Doct. 34:198-200.[Medline]
17.	Kirby, A. C., M. Sundquist, and M. J. Wick. 2004. In vivo compartmentalization of functionally distinct, rapidly responsive antigen-specific T-cell populations in DNA-immunized or Salmonella enterica serovar Typhimurium-infected mice. Infect. Immun. 72:6390-6400.[Abstract/Free Full Text]
18.	Ley, R. E., D. A. Peterson, and J. I. Gordon. 2006. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124:837-848.[CrossRef][Medline]
19.	Lo, W. F., H. Ong, E. S. Metcalf, and M. J. Soloski. 1999. T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules. J. Immunol. 162:5398-5406.[Abstract/Free Full Text]
20.	Luu, R. A., K. Gurnani, R. Dudani, R. Kammara, H. van Faassen, J. C. Sirard, L. Krishnan, and S. Sad. 2006. Delayed expansion and contraction of CD8+ T cell response during infection with virulent Salmonella typhimurium. J. Immunol. 177:1516-1525.[Abstract/Free Full Text]
21.	Lyons, A. B., and C. R. Parish. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171:131-137.[CrossRef][Medline]
22.	Macpherson, A. J., and T. Uhr. 2004. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 303:1662-1665.[Abstract/Free Full Text]
23.	Mastroeni, P., and N. Menager. 2003. Development of acquired immunity to Salmonella. J. Med. Microbiol. 52:453-459.[Abstract/Free Full Text]
24.	Mastroeni, P., C. Simmons, R. Fowler, C. E. Hormaeche, and G. Dougan. 2000. Igh-6–/– (B-cell-deficient) mice fail to mount solid acquired resistance to oral challenge with virulent Salmonella enterica serovar Typhimurium and show impaired Th1 T-cell responses to Salmonella antigens. Infect. Immun. 68:46-53.[Abstract/Free Full Text]
25.	Mastroeni, P., B. Villarreal-Ramos, and C. E. Hormaeche. 1992. Role of T cells, TNF alpha and IFN in recall of immunity to oral challenge with virulent salmonellae in mice vaccinated with live attenuated aro– Salmonella vaccines. Microb. Pathog. 13:477-491.[CrossRef][Medline]
26.	McCollister, B. D., T. J. Bourret, R. Gill, J. Jones-Carson, and A. Vazquez-Torres. 2005. Repression of SPI2 transcription by nitric oxide-producing, IFN-activated macrophages promotes maturation of Salmonella phagosomes. J. Exp. Med. 202:625-635.[Abstract/Free Full Text]
27.	Miao, E. A., M. Brittnacher, A. Haraga, R. L. Jeng, M. D. Welch, and S. I. Miller. 2003. Salmonella effectors translocated across the vacuolar membrane interact with the actin cytoskeleton. Mol. Microbiol. 48:401-415.[CrossRef][Medline]
28.	Miao, E. A., and S. I. Miller. 2000. A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 97:7539-7544.[Abstract/Free Full Text]
29.	Mittrucker, H. W., and S. H. Kaufmann. 2000. Immune response to infection with Salmonella typhimurium in mice. J. Leukoc. Biol. 67:457-463.[Abstract]
30.	Mittrucker, H. W., B. Raupach, A. Kohler, and S. H. Kaufmann. 2000. Cutting edge: role of B lymphocytes in protective immunity against Salmonella typhimurium infection. J. Immunol. 164:1648-1652.[Abstract/Free Full Text]
31.	Monack, D. M., D. M. Bouley, and S. Falkow. 2004. Salmonella typhimurium persists within macrophages in the mesenteric lymph nodes of chronically infected Nramp1+/+ mice and can be reactivated by IFN neutralization. J. Exp. Med. 199:231-241.[Abstract/Free Full Text]
32.	Mora, J. R., M. R. Bono, N. Manjunath, W. Weninger, L. L. Cavanagh, M. Rosemblatt, and U. H. Von Andrian. 2003. Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells. Nature 424:88-93.[CrossRef][Medline]
33.	O'Brien, R. L., C. E. Roark, C. Reardon, K. Heyborne, and W. Born. 1997. Chapter 21.4, p. 1503-1508. In I. Lefkovits (ed.), Immunology methods manual: the comprehensive sourcebook of techniques, vol. 4. Academic Press, New York, NY.
34.	Rescigno, M., M. Urbano, B. Valzasina, M. Francolini, G. Rotta, R. Bonasio, F. Granucci, J. P. Kraehenbuhl, and P. Ricciardi-Castagnoli. 2001. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol. 2:361-367.[CrossRef][Medline]
35.	Russmann, H., H. Shams, F. Poblete, Y. Fu, J. E. Galan, and R. O. Donis. 1998. Delivery of epitopes by the Salmonella type III secretion system for vaccine development. Science 281:565-568.[Abstract/Free Full Text]
36.	Salerno-Goncalves, R., R. Wahid, and M. B. Sztein. 2005. Immunization of volunteers with Salmonella enterica serovar Typhi strain Ty21a elicits the oligoclonal expansion of CD8+ T cells with predominant Vß repertoires. Infect. Immun. 73:3521-3530.[Abstract/Free Full Text]
37.	Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708-712.[CrossRef][Medline]
38.	Sprent, J. 2003. Turnover of memory-phenotype CD8+ T cells. Microbes Infect. 5:227-231.[CrossRef][Medline]
39.	Svensson, M., B. Stockinger, and M. J. Wick. 1997. Bone marrow-derived dendritic cells can process bacteria for MHC-I and MHC-II presentation to T cells. J. Immunol. 158:4229-4236.[Abstract]
40.	Tsolis, R. M., A. J. Baumler, I. Stojiljkovic, and F. Heffron. 1995. Fur regulon of Salmonella typhimurium: identification of new iron-regulated genes. J. Bacteriol. 177:4628-4637.[Abstract/Free Full Text]
41.	Tsukada, C., C. Miyaji, H. Kawamura, R. Miyakawa, H. Yokoyama, Y. Ishimoto, S. Miyazawa, H. Watanabe, and T. Abo. 2003. Characterization of extrathymic CD8 alpha beta T cells in the liver and intestine in TAP-1 deficient mice. Immunology 109:343-350.[CrossRef][Medline]
42.	van der Velden, A. W., M. K. Copass, and M. N. Starnbach. 2005. Salmonella inhibit T cell proliferation by a direct, contact-dependent immunosuppressive effect. Proc. Natl. Acad. Sci. USA 102:17769-17774.[Abstract/Free Full Text]
43.	Van Kaer, L., P. G. Ashton-Rickardt, H. L. Ploegh, and S. Tonegawa. 1992. TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4-8+ T cells. Cell 71:1205-1214.[CrossRef][Medline]
44.	Vazquez-Torres, A., and F. C. Fang. 2000. Cellular routes of invasion by enteropathogens. Curr. Opin. Microbiol. 3:54-59.[CrossRef][Medline]
45.	Vazquez-Torres, A., J. Jones-Carson, A. J. Baumler, S. Falkow, R. Valdivia, W. Brown, M. Le, R. Berggren, W. T. Parks, and F. C. Fang. 1999. Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes. Nature 401:804-808.[CrossRef][Medline]
46.	Wherry, E. J., and R. Ahmed. 2004. Memory CD8 T-cell differentiation during viral infection. J. Virol. 78:5535-5545.[Free Full Text]
47.	Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. von Andrian, and R. Ahmed. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4:225-234.[CrossRef][Medline]
48.	Whitman, W. B., D. C. Coleman, and W. J. Wiebe. 1998. Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. USA 95:6578-6583.[CrossRef][Medline]
49.	Wijburg, O. L., N. Van Rooijen, and R. A. Strugnell. 2002. Induction of CD8+ T lymphocytes by Salmonella typhimurium is independent of Salmonella pathogenicity island 1-mediated host cell death. J. Immunol. 169:3275-3283.[Abstract/Free Full Text]

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