Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Psychoneuroimmunology wikipedia , lookup
Immune system wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Molecular mimicry wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Adaptive immune system wikipedia , lookup
This information is current as of June 18, 2017. Massive Number of Antigen-Specific CD4 T Cells during Vaccination with Live Attenuated Salmonella Causes Interclonal Competition Aparna Srinivasan, Joseph Foley and Stephen J. McSorley J Immunol 2004; 172:6884-6893; ; doi: 10.4049/jimmunol.172.11.6884 http://www.jimmunol.org/content/172/11/6884 Subscription Permissions Email Alerts This article cites 44 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/172/11/6884.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References The Journal of Immunology Massive Number of Antigen-Specific CD4 T Cells during Vaccination with Live Attenuated Salmonella Causes Interclonal Competition1 Aparna Srinivasan, Joseph Foley, and Stephen J. McSorley2 The clonal burst size of CD4 T cells is predicted to be less than that of CD8 T cells. In this study, we demonstrate that massive numbers of Ag-specific CD4 T cells respond during vaccination of mice with live attenuated Salmonella, reaching a peak frequency of ⬃50% of CD4 T cells. Salmonella-specific T cells persisted at high frequency for several weeks and could be detected in the memory population for months after infection. Surprisingly, the expansion of endogenous Salmonella-specific CD4 T cells prevented the persistence of adoptively transferred Salmonella-specific T cells in vivo, demonstrating interclonal competition for access to the memory compartment. The Journal of Immunology, 2004, 172: 6884 – 6893. to the notion that the clonal burst size of CD4 T cells is restricted, perhaps due to inefficient Ag presentation of class-II restricted epitopes, or an intrinsic defect in the proliferative program of CD4 cells (11). Indeed, studies using an adoptive transfer model where Listeria-specific TCR-transgenic CD4 and CD8 cells could be directly visualized in vivo noted a clear difference in the proliferative capacity of these two populations (21). However, it is not clear whether this represents a general phenomenon for all CD4 T cells or is only true in models of infection where CD8 effector cells are critical for pathogen clearance. The resolution of Salmonella enterica serovar typhimurium (hereafter referred to as Salmonella typhimurium) infection is critically dependent upon the activation of CD4 T cells and the production of IFN-␥ to activate infected macrophages (22, 23). Furthermore, live vaccine strains of Salmonella induce robust CD4-dependent protective immunity in susceptible mice (24). We reasoned that this might be a more attractive model to examine the clonal burst size of CD4 T cells in response to infection. In this study, we report that a massive number of endogenous Salmonella-specific CD4 T cells are expanded following vaccination with live attenuated Salmonella, reaching a peak at around 50% of all CD4 T cells. Salmonella-specific CD4 cells are maintained at this high frequency until the bacteria are eliminated and can be detected in the CD4 memory pool for months after vaccination. We also demonstrate that the massive number of endogenous Salmonella-specific CD4 T cells hinders the survival of Salmonella flagellin-specific CD4 T cells, demonstrating interclonal competition for access to the memory CD4 compartment. Materials and Methods Department of Medicine, Division of Immunology, University of Connecticut Health Center, Farmington, CT 06030 Mouse strains Received for publication January 23, 2004. Accepted for publication March 19, 2004. SM1 Rag-deficient TCR-transgenic mice expressing the Thy1.1 or CD45.1 allele were produced by backcrossing the original C57BL/6 SM1 Ragdeficient line (25) to B6.PL-thy1a/Cy or B6.SJL-PtprcaPep3b/BoyJ mouse strains (The Jackson Laboratory, Bar Harbor, ME). OT-II TCR-transgenic mice (26) were generously provided by Dr. L. Lefrancois (University of Connecticut Health Center). C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and used at 8 –16 wk of age. All mice were housed in specific pathogen-free conditions and cared for in accordance with the University of Connecticut Health Center and National Institutes of Health guidelines. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from National Institutes of Health (AI056172 and AI055743 to S.J.M) and the Robert Leet and Clara Guthrie Patterson Trust (to S.J.M.). 2 Address correspondence and reprint requests to Dr. Stephen J. McSorley, Department of Medicine, Division of Immunology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030. E-mail address: [email protected] Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 T he theory of clonal selection (1) predicts a low frequency of Ag-specific T cell clones, and this has been confirmed experimentally (2, 3). A rapidly dividing pathogen could potentially exploit such an inherent frailty in the adaptive immune response. Therefore, a successful immune response depends upon the rapid expansion of pathogen-specific clones that express appropriately rearranged Ag receptors. After pathogen clearance, the maintenance of these clones at an elevated frequency constitutes an important aspect of immunological memory (4). Extensive proliferation of Ag-specific CD8 clones has been reported following viral infection, comprising 50 –70% of all CD8 cells at the peak of the response (5, 6). Contraction of the virusspecific response then follows, leaving an elevated frequency of Ag-specific cells in the memory pool. The burst size of a given virus-specific population correlates with frequency in the memory pool, suggesting that there is minimal interclonal competition between responding CD8 T cells (5). Some studies have suggested that competition between CD8 clones of different Ag specificity can occur in vivo (7–9), while others support the idea that competition primarily occurs between cells of identical specificity (6, 10). Although a great deal has been learned about the nature of the CD8 response to viral infection, it is not clear whether the same rules apply to pathogen-specific CD4 T cells (11). Numerous studies have reported that the frequency of Ag-specific CD8 T cells substantially exceeds that of Ag-specific CD4 T cells at the peak of the immune response (12–15). Experiments using in vitro restimulation assays have estimated that around 2–3% of CD4 cells are Ag specific at the peak of the response to viral infection (12, 16 –19), although studies with lymphocytic choriomeningitis virus have suggested it could be as high as 10% (13, 20). These data have led The Journal of Immunology Adoptive transfer of SM1 T cells Spleen and lymph node cells (inguinal, axillary, brachial, cervical, mesenteric, periaortic) were harvested from SM1 RAG-deficient, CD90.1 congenic (or CD45.1 congenic) TCR-transgenic mice, and a pooled single-cell suspension was generated. A small aliquot of cells was stained using Abs to CD4, CD90.1 (or CD45.1), and V2 (BD Biosciences, San Diego, CA) and the percentage of SM1 T cells was determined by flow cytometry using a FACSCalibur (BD Biosciences). Cell numbers were adjusted accordingly and 2–5 ⫻ 106 SM1 T cells were injected i.v. into recipient C57BL/6 or OT-II mice. In most experiments, SM1 cells were also stained with CFSE (27) before adoptive transfer. Vaccination with live attenuated Salmonella or heat-killed S. typhimurium (HKST)3 immunity. It is possible that the CD4 burst size is far greater in models where CD4 T cells are the primary effector cell mediating pathogen clearance. Vaccination of C57BL/6 mice with attenuated S. typhimurium provides protective immunity to challenge with virulent bacteria that is dependent upon CD4 T cells (24). In initial experiments, we noted that the total number of splenocytes increased markedly 1 wk after vaccination with attenuated Salmonella, largely due to an increase in the number of CD11b- and Gr1-expressing non-T cells (Fig. 1A and data not shown). Although this splenomegaly decreased the percentage of CD4 and CD8 T cells (Fig. 1B), the absolute number of splenic CD4 and CD8 T cells actually increased ⬃2-fold in the infected spleen (Fig. 1C). Phenotypic analysis of the CD4 population demonstrated that this 2-fold increase was due to a large increase in the number of cells with an activated phenotype, CD4⫹CD11ahighCD44⫹ (Fig. 1D and data not shown). Furthermore, a sizable percentage of CD4 T cells from vaccinated mice produced IFN-␥ and TNF-␣ directly ex vivo (Fig. 1D). CD4 cells from vaccinated mice did not produce IL-4 or IL-10 (data not shown), although a small proportion produced IL-2 (Fig. 1D). All cytokine production by CD4 cells was confined to the CD4⫹CD11ahigh population (Fig. 1E). A number of non-CD4, Flow cytometric analysis Spleen or lymph node cells were incubated on ice for 20 – 45 min in Fc block (spent culture supernatant from the 24G2 hybridoma, 2% rat serum, 2% mouse serum, and 0.01% sodium azide) in the presence of the relevant primary Abs. FITC-, PE-, CyChrome-, PE-Cy5-, allophycocyanin-, or biotin-conjugated Abs specific for CD4, CD11a, CD45.1, CD69, CD90.1, IL-2, IL-4, IL-10, TNF-␣, IFN-␥, V2, 3, 4, 5.1/5.2, 6, 7, 8.1/8.2, 8.3, 9, 10b, 11, 12, 13, 14, 17a, and isotype control Abs were purchased from BD PharMingen (San Diego, CA). Following staining, cells were analyzed by flow cytometry using a FACSCalibur. Data were analyzed using FlowJo software (TreeStar, San Carlos, CA). Tracking SM1 T cells and Salmonella At various times after infection, spleen or lymph node cells were harvested in Eagle’s Hanks’ amino acids medium (Biofluids, Rockville, MD) containing 2% FBS and 5 mM EDTA. Serial dilutions of each sample were plated onto MacConkey agar plates (Difco, Detroit, MI) at 37°C to determine bacterial colonization in each tissue. Cells from the same organ (5 ⫻ 106/tube) were then processed and stained as described above. In vivo cytokine stimulation and intracellular staining For intracellular cytokine experiments examining SM1 T cells, mice were injected i.v. with 200 g of flagellin peptide 427– 441 (VQNRFNSAIT NLGNT) (29) plus 25 g of LPS (Sigma-Aldrich, St. Louis, MO). For intracellular cytokine experiments examining endogenous Salmonella-specific CD4 T cells, mice were injected i.v. with 1 ⫻ 108 HKST. To examine cytokine production directly ex vivo, spleens from such injected mice were harvested at 2 h after injection. Spleen cells were rapidly surface stained, fixed with formaldehyde, permeabilized using saponin (Sigma-Aldrich), and stained intracellularly using the Abs described above. Results Vaccination with live Salmonella increases the number of CD4 memory cells in the spleen Although a number of studies have examined the clonal burst size of CD4 cells (12–21), these have used viral or bacterial models where CD8 T cell expansion is likely to be required for protective 3 Abbreviations used in this paper: HKST, heat-killed Salmonella typhimurium; HKLM, heat-killed Listeria monocytogene; SAg, superantigen. FIGURE 1. Vaccination with live Salmonella causes a large expansion of activated CD4 T cells in vivo. C57BL/6 mice were vaccinated with 5 ⫻ 105 live attenuated Salmonella (vaccinated) or were left untreated (naive). One week later, spleen cells were stained and the total cell number (A), percentage of CD4 cells (B), total number of CD4 cells (C) were determined. Data show the mean ⫾ SD of three mice per group and are representative of four individual experiments. D, The surface expression of CD11a and the production of intracellular cytokines were examined in CD4 cells from the spleen of naive and vaccinated mice. Data show representative plots of spleen cells from at least three individual mice. Numbers indicate the percentage of spleen cells within the boxed gate. E, CD11a and intracellular staining after gating on live CD4 T cells from naive or infected mice. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Attenuated (AroA⫺D⫺) S. typhimurium (BRD509) (28) was provided by Dr. D. Xu (University of Glasgow, Glasgow, U.K.). Flagellin-deficient S. typhimurium (BC490) was generously provided by Dr. B. Cookson (University of Washington, Seattle, WA). Bacteria were routinely grown overnight in Luria-Bertani broth without shaking, and diluted in PBS after an estimation of bacterial concentration using a spectrophotometer. Aliquots of live bacterial suspensions were incubated at 75°C for 1 h to generate Heat-killed S. typhimurium (HKST). Live attenuated (5 ⫻ 105) and heatkilled bacteria (1 ⫻ 108), were administered i.v. into the lateral tail vein of mice. In all experiments, the live bacterial dose was confirmed by serial plating onto MacConkey agar plates. In preliminary experiments, the challenge doses chosen for this study were found to generate similar peak SM1 clonal expansion and were therefore chosen for further study. In some experiments, the recall response of SM1 T cells to flagellin peptide immunization was examined in immunized mice. Adoptively transferred and previously immunized mice were injected i.v. with 200 g flagellin peptide and 25 g LPS and clonal expansion of SM1 T cells was examined 3 days later. 6885 6886 FIGURE 2. Kinetics of CD4 CD11a⫹ and IFN-␥⫹ expansion after vaccination with attenuated Salmonella. C57BL/6 mice were vaccinated with 5 ⫻ 105 live attenuated Salmonella and the number of CD4 cells, CD11a⫹CD4 cells, and IFN-␥⫹CD4 cells (A) or number of viable Salmonella (B) determined in the spleen at various time points later. C, CD11apositive and IFN-␥- positive CD4 cells expressed as a percentage of all CD4 cells in the spleen of infected mice. Data show the mean ⫾ SD of two to four mice per time point. Most CD4⫹CD11ahigh T cells in the spleen are Salmonella specific These data suggested that the Salmonella burden in the spleen could be directly responsible for the specific activation of a large number of CD4 T cells to produce IFN-␥ ex vivo. However, even if this were true, it still would not account for the vast majority of the expanded CD4⫹CD11ahigh population found in the spleen after live vaccination. IFN-␥-producing CD4 cells represent ⬍20% of the total CD4⫹CD11ahigh population during the response to live vaccination (Fig. 2C). From these data, it was not possible to distinguish whether non-IFN-␥-producing CD4⫹CD11ahigh cells represented Salmonella-specific CD4 T cells that had not been recently activated in vivo, or were simply CD4⫹CD11high T cells that were not specific for Salmonella. To discriminate between these possibilities, we modified an in vivo peptide pulse assay that has been used to examine the cytokine production of TCR-transgenic CD4 T cells in vivo (30, 31). This assay typically involves the i.v. injection of peptide to synchronously activate TCR-transgenic T cells in vivo, followed by analysis of intracellular cytokine production ex vivo, only 2 h later. Although the Ag specificity of the endogenous CD4 response to Salmonella is unknown, we hypothesized that most target Ags would be present in a HKST Ag preparation. C57BL/6 mice were therefore vaccinated with live attenuated Salmonella and, 1 wk later, some mice were injected with HKST. Two hours after this HKST pulse, spleens were harvested and rapidly processed to examine intracellular cytokine production. As in our previous experiments, an increased frequency of IFN-␥- and TNF-␣-producing CD4 T cells was detected in vaccinated mice (Fig. 3A, naive vs vaccinated). These cytokine-producing cells were only found in the CD4⫹CD11ahigh population (Fig. 3B). A very small increase in CD4 IFN-␥ and TNF-␣ production was detected in naive C57BL/6 mice that had received the HKST pulse (Fig. 3A, naive ⫹ HKST), and these cells also expressed CD11ahigh. It is possible that these cells are cross-reactive CD4 memory cells specific for the endogenous bacterial flora, but we have not examined this hypothesis in detail. A massive increase in the fraction of CD4 T cells producing IFN-␥ and the amount of IFN-␥ per cell was detected in vaccinated mice that had been pulsed with HKST (Fig. 3A, vaccinated ⫹ HKST). Again, this cytokine response only occurred in the CD11ahigh population and actually accounted for the vast majority of all CD4⫹CD11ahigh T cells (Fig. 3B). It seemed likely that the HKST pulse assay allowed for the identification of Salmonella-specific T cells and that these cells accounted for a surprisingly high fraction of the CD4 population, in mice that were vaccinated with live Salmonella. However, it was possible that the response to the HKST pulse was either a nonspecific inflammatory response or a specific response to an unknown Salmonella superantigen (SAg). To address the latter concern, we analyzed the TCR V repertoire of CD4 T cells producing IFN-␥ and those not producing IFN-␥ after the HKST pulse, as SAg responses involve the restricted activation of certain V-expressing T cells (32). Although a 2-fold increase in V9-, 12-, and 17a-expressing CD4 T cells was noted among IFN-␥positive cells, there was no obvious bias in the V repertoire that would indicate a SAg response (Fig. 3C). To examine the effect of nonspecific bacterial inflammation on CD4 IFN-␥ production, we pulsed vaccinated mice with HKST or heat-killed Listeria monocytogenes (HKLM). Although HKST induced CD4 IFN-␥ production, pulsing vaccinated mice with HKLM did not increase the frequency of IFN-␥-producing CD4 cells detected in the spleen (Fig. 3D and data not shown). We conclude that the in vivo pulse Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 non-CD8 cells in vaccinated mice stained positive with all Abs, including isotype control Abs (Fig. 1D). This nonspecific staining is likely to result from the increased number of CD11b⫹ cells in the spleen of vaccinated mice. We examined the in vivo kinetics of the CD4⫹CD11ahigh response. Vaccination with live Salmonella caused a transient increase in total splenic CD4 numbers that had largely dissipated 2 mo later (Fig. 2A, total CD4 cells). The increase in CD4 numbers was due to a large expansion of the CD4⫹CD11ahigh T cell population (Fig. 2A, total CD11a⫹ CD4 cells). This kinetics of the CD4⫹CD11ahigh response had three distinct phases: 1) an expansion phase between day 0 and days 7–14, when the absolute number of these cells increased ⬃10-fold; 2) a plateau until days 21– 28, when the number of these cells remained relatively constant at a high level; 3) a contraction phase after days 21–28, when the number of cells declined steadily (Fig. 2A). We also examined the kinetics of the CD4⫹IFN-␥⫹ response, the magnitude and kinetics of which was slightly different (Fig. 2A, total IFN-␥⫹ CD4 cells). An increased frequency of CD4⫹IFN␥⫹-producing cells was detected as early as 3 days after live vaccination. This response peaked at around 1 wk and stayed relatively constant until 3 wk following vaccination, after which it disappeared completely. Interestingly, the kinetics of the IFN-␥producing CD4 cell response closely mirrored the kinetics of the bacterial burden in the spleen (Fig. 2B). Numbers of Salmonella increased during the first week, stayed at relatively high levels for 2–3 wk, and then rapidly disappeared. CD4 T CELL RESPONSE TO LIVE ATTENUATED Salmonella The Journal of Immunology 6887 assay allows the detection of endogenous Salmonella-specific CD4 T cells in vivo and that these cells comprise a surprisingly large fraction of the CD4 population in vaccinated mice. Live vaccination causes a massive increase in Salmonella-specific CD4 T cells but vaccination with HKST does not We used the HKST pulse assay to examine the frequency and kinetics of the endogenous Salmonella-specific CD4 response. These data demonstrate that a huge clonal expansion of Salmonella-specific CD4 T cells occurred in the first week after live vac- cination (Fig. 4A, total IFN-␥⫹ CD4 cells). The number of these cells persisted at a very high level for 3– 4 wk, after which there was a significant decline. Importantly, the kinetics of IFN-␥-producing cells closely mirrored the kinetics of the CD11a⫹ population in response to vaccination (Fig. 4A). Indeed, it was possible to account for all of the increase in the CD4⫹CD11ahigh population as being due to an increase in Salmonella-specific CD4 T cells (Fig. 4B). At the peak of the response, Salmonella-specific CD4 T cells accounted for ⬃50% of all CD4 T cells (Fig. 4B). Among Salmonella-specific CD4 cells, ⬃30 – 40% also produced TNF-␣ (Fig. 4B) and ⬃5% produced IL-2 (data not shown). No production of Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 3. Pulsing infected mice with HKST allows the identification of endogenous Salmonella-specific CD4 T cells in vivo. A, Groups of C57BL/6 mice were vaccinated with 5 ⫻ 105 live attenuated Salmonella and 1 wk later some mice were injected i.v. with 1 ⫻ 108 HKST. Spleens were harvested 2 h later and intracellular production of IFN-␥ and TNF-␣ by CD4 T cells was determined. Data show cytokine production among live spleen cells and are representative of two to three mice per group and four individual experiments. Numbers represent the percentage of spleen cells falling within the boxed gate. B, CD11a expression and cytokine production from the same groups after gating on CD4 T cells. C, Groups of C57BL/6 mice were vaccinated with 5 ⫻ 105 live attenuated Salmonella and 3 wk later injected i.v. with 1 ⫻ 108 HKST. Spleens were harvested 2 h later and intracellular production of IFN-␥ and expression of TCR V receptors were determined among CD4 T cells. IFN-␥-negative and -positive cells were defined using a boxed gate similar to that shown in A. Data show the percentage of CD4 cells expressing a particular V chain after gating on IFN-␥-positive or -negative CD4 cells. D, Groups of C57BL/6 mice were vaccinated with 5 ⫻ 105 live attenuated Salmonella and 1 wk later some mice were injected i.v. with 1 ⫻ 108 HKST or 1 ⫻ 108 HKLM. Spleens were harvested 2 h later and intracellular production of IFN-␥ was determined. Numbers represent the percentage of spleen cells falling within the boxed gate. 6888 nodes and axillary-brachial-linguinal (ABI) nodes of C57BL/6 mice after adoptive transfer (Fig. 5). In response to live vaccination, SM1 T cells expanded in the spleen but not in the mesenteric lymph nodes or peripheral ABI lymph nodes (Fig. 5). This restricted activation of SM1 T cells is reminiscent of our previous observation that SM1 T cell expansion is limited to the gutassociated lymphoid tissue following oral Salmonella infection (25) and likely reflects an intrinsic property of the immune response to Salmonella flagellin. However, the most surprising finding was that by 10 days after vaccination very few SM1 T cells could be detected in the spleen or any other lymphoid tissue examined (Fig. 5). Therefore, despite the massive clonal expansion of adoptively transferred SM1 T cells at days 3–5, they are not found at an elevated frequency at later time points. Indeed, by day 10, frequencies of SM1 T cells typically drop below the low level detected in transfer only mice (Fig. 5). It was possible that the unusual inability to generate SM1 memory cells was an intrinsic property of SM1 T cells or of flagellinspecific T cells in general. We therefore attempted to generate memory SM1 T cells after immunization with HKST or flagellin peptide and LPS. After immunization with HKST or peptide/LPS, SM1 T cells expanded in the spleen, mesenteric lymph nodes, and ABI lymph nodes (Fig. 5 and data not shown). By day 10 or 20 after immunization, an increased frequency of SM1 T cells was detected in all lymphoid tissues analyzed (Fig. 5 and data not shown). In some experiments, the peak expansion of SM1 cells to HKST was greater than that of live vaccination (Fig. 5B). However, this did not explain the ability of these cells to persist in vivo. The relative decrease from the peak frequency was always severalfold greater following live vaccination than after HKST vaccination. Thus, the inability to generate an elevated frequency of IL-4 or IL-10 by CD4 T cells was detected at any time point using this assay (data not shown). We examined whether this expansion of Salmonella-specific CD4 cells could be observed after vaccination with Salmonella Ags or whether it only occurred following vaccination with live bacteria. C57BL/6 mice were immunized i.v. with HKST and 6 and 11 days later, IFN-␥ and TNF-␣ production of CD4 T cells was examined using the HKST pulse assay exactly as described above. CD4 T cells in the spleen of HKST-vaccinated mice did not produce large quantities of cytokines in response to the in vivo pulse (Fig. 4C and data not shown) Salmonella-specific TCR-transgenic cells do not develop into memory cells following vaccination with live Salmonella We sought to examine the clonal expansion and development of Salmonella-specific memory CD4 cells in more detail using an adoptive transfer approach. We previously generated the SM1 TCR-transgenic mouse specific for Salmonella flagellin (25), and therefore decided to use this model to examine the CD4 response to live vaccination. A small population of SM1 T cells (CD4⫹CD90.1⫹) was detected in the spleen, mesenteric lymph FIGURE 5. SM1 T cells do not persist after live attenuated Salmonella vaccination. C57BL/6 mice were adoptively transferred with 2 ⫻ 106 SM1 T cells and groups of mice were vaccinated the following day with 5 ⫻ 105 live attenuated Salmonella or 1 ⫻ 108 HKST. A, Spleen cells were harvested 5 and 10 days later and the percentage of SM1 T cells was determined. Plots are representative of three mice per group and three similar experiments. B, In another similar experiment, spleen, mesenteric lymph nodes, and ABI lymph nodes were harvested at various time points following vaccination with live Salmonella or HKST and the percentage of SM1 T cells in each tissue was determined by flow cytometry. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 4. Massive expansion of Salmonella-specific CD4 T cells after vaccination with live Salmonella but not HKST. A, C57BL/6 mice were vaccinated with 5 ⫻ 105 live attenuated Salmonella and injected with 1 ⫻ 108 HKST at various time points later. Two hours after HKST injection, spleen cells were harvested and the number of CD4 cells, CD11a⫹ CD4 cells, and IFN-␥⫹ CD4 cells was determined. Data show the mean ⫾ SD of two to four mice per time point. B, CD11a-positive and IFN-␥-positive CD4 cells expressed as a percentage of all CD4 cells in the spleen of infected mice. C, C57BL/6 mice were vaccinated with 1 ⫻ 108 HKST and 11 days later some mice were injected with 1 ⫻ 108 HKST to stimulate cytokine production. Two hours later, spleen cells were harvested and cytokine production of CD4 T cells was examined by intracellular staining. The number of CD4 cells, CD11a⫹ CD4 cells, and IFN-␥⫹ CD4 cells was determined. Numbers indicate the percentage of spleen cells within the boxed gate. CD4 T CELL RESPONSE TO LIVE ATTENUATED Salmonella The Journal of Immunology 6889 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 6. SM1 T cells are functionally responsive to peptide in mice administered live Salmonella. C57BL/6 mice were adoptively transferred with 2 ⫻ 106 CFSE-labeled SM1 T cells and groups of mice were vaccinated the following day with 5 ⫻ 105 live attenuated Salmonella or 1 ⫻ 108 HKST. A, Spleen cells were harvested 5 days later and CFSE staining was examined by flow cytometry after gating on CD4⫹CD90.1⫹ SM1 T cells. Data are representative of three individual mice and two separate experiments. B, C57BL/6 mice were adoptively transferred with 2 ⫻ 106 CFSE-labeled SM1 T cells and groups of mice were vaccinated the following day with 5 ⫻ 105 live attenuated Salmonella or 1 ⫻ 108 HKST. Five days after vaccination, some mice were injected i.v. with 200 g of flagellin peptide and 25 g of LPS (⫹ peptide), and some mice were left untreated (⫺ peptide). Two hours after peptide injection, spleen cells were harvested and stained using various different Abs. Data show surface staining for CD69 and intracellular staining for IL-2 or IFN-␥ after gating on splenic CD4⫹CD90.1⫹ SM1 T cells. Plots from individual mice are shown and are representative of two individual mice and two individual experiments. Numbers represent the percentage of SM1 T cells in the boxed gate. C, C57BL/6 mice were adoptively transferred with 2 ⫻ 106 SM1 T cells and groups of mice were vaccinated the following day with 5 ⫻ 105 live attenuated Salmonella or 1 ⫻ 108 HKST. Two weeks later, groups of mice were injected i.v. with 200 g of flagellin peptide and LPS. Three days later, spleen cells were harvested and the percentage of SM1 T cells was determined. Numbers represent the percentage of SM1 T cells in the spleen, as defined by the boxed gate. 6890 CD4 T CELL RESPONSE TO LIVE ATTENUATED Salmonella SM1 T cells is a specific property of live vaccination and is not due to an intrinsic defect in SM1 TCR-transgenic cells. Vaccination with live Salmonella or HKST induces SM1 T cell populations with similar phenotypic and functional properties Endogenous Salmonella-specific T cells prevent the development of SM1 memory cells Our data suggested that a massive expansion and maintenance of endogenous Salmonella-specific T cells occurs in response to live FIGURE 7. Salmonella-specific endogenous CD4 T cells hinder the persistence of SM1 T cells in vivo. A, C57BL/6 or OT-II mice were adoptively transferred with CFSE-labeled 2 ⫻ 106 SM1 T cells and groups of mice were vaccinated the following day with 5 ⫻ 105 live attenuated Salmonella. Spleen cells were harvested 3 and 10 days later, and the percentage of SM1 T cells was determined. CFSE plots are gated only on SM1 T cells, as defined by the boxed gate shown. Numbers represent the percentage of SM1 T cells in the spleen. B, Groups of C57BL/6 mice were vaccinated with 5 ⫻ 105 live attenuated Salmonella and 1 wk later some mice were injected i.v. with 1 ⫻ 108 HKST prepared from flagellated (HKST) or aflagellate bacteria (aflagellate HKST). Spleens were harvested 2 h later and intracellular production of IFN-␥ was determined. Data show the percentage of CD4 T cells producing IFN-␥ as a percentage of all CD4 T cells in the spleen. vaccination. In contrast, SM1 T cells expand but are unable to survive at later time points. We therefore examined the possibility that these two populations of cells compete with each other during live vaccination. We decided to examine SM1 T cell persistence in a model where endogenous CD4 T cell competition cannot occur in vivo. SM1 T cells were therefore adoptively transferred into C57BL/6 mice or OT-II TCR-transgenic mice. All endogenous CD4 T cells in the periphery of OT-II mice are specific for OVA and should be unable to respond during vaccination with live Salmonella. Three days following live Salmonella vaccination, bacterial counts in the spleen of both recipient groups were similar (data not shown). SM1 T cells in both C57BL/6 and OT-II recipients expanded in response to vaccination, increased expression of CD11a, and underwent multiple rounds of cell division, as observed by CFSE staining (Fig. 7A, day 3). SM1 T cells in OT-II mice expanded slightly more than those in C57BL/6 mice. As in previous experiments, SM1 T cells did not persist in the spleen of C57BL/6 mice vaccinated with live Salmonella (Fig. 7A, day 10). Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Immunizing mice with live Salmonella or HKST generated a similar frequency of splenic SM1 cells on day 5, but a very different frequency by day 10. To understand this difference, we examined the phenotypic and functional properties of SM1 T cells at day 5. Analysis of later time points was difficult due to the rapid loss of the SM1 population in mice vaccinated with live Salmonella (Fig. 5). On day 5, SM1 cells in the spleen of both groups of mice had undergone multiple rounds of cell division and increased surface expression of CD11a (Fig. 6A) and CD44 (data not shown). Recent reports suggest that IFN-␥-producing CD4 T cells may be short-lived and unable to develop into memory T cells (33). It was possible that live Salmonella induce a more pronounced IFN-␥ response in SM1 T cells, leading to their inability to survive at later time points. We therefore examined the cytokine production of SM1 T cells directly ex vivo after vaccination with live Salmonella or HKST. Five days after vaccination, SM1 T cells were uniformly CD69low, IL-2⫺, and IFN-␥⫺ (Fig. 6B). We next sought to examine the functional response of these SM1 T cells after in vivo stimulation with flagellin peptide and LPS. Five days after vaccination, transfer only, live Salmonella, or HKST mice were injected i.v. with peptide/LPS to activate SM1 cells. Intracellular cytokine production was examined 2 h later. SM1 T cells in transfer only, live Salmonella, or HKST mice increased surface expression of CD69 in response to in vivo peptide stimulation, indicating that all of these cells encountered peptide (Fig. 6B). Approximately 20% of SM1 T cells transfer-only mice produced IL-2 in response to peptide stimulation, and this was increased in mice exposed to live Salmonella or HKST (Fig. 6B). Very few naive SM1 cells in transfer-only mice produced IFN-␥ in response to peptide stimulation (Fig. 6B). A higher percentage of SM1 cells produced IFN-␥ in mice administered live Salmonella or HKST, but there was no noticeable difference between the vaccination regimens (Fig. 6B). These data indicate that SM1 T cells in mice immunized with HKST or live Salmonella are functionally similar and that excess IFN-␥ production is unlikely to explain the loss of SM1 T cells observed at later time points. Next, we examined whether the few SM1 T cells that remain at later time points in mice vaccinated with live Salmonella are actually functionally responsive. C57BL/6 mice were adoptively transferred with SM1 T cells and vaccinated with HKST or live Salmonella or were left unimmunized (transfer only). Two weeks later, some mice were immunized i.v. with flagellin peptide/LPS and clonal expansion of SM1 T cells was examined 3 days later. A small population of SM1 T cells was detected in the spleen of transfer-only mice, and these cells expanded in response to peptide/LPS stimulation (Fig. 6C). In agreement with previous experiments, HKST-immunized mice had a higher frequency of SM1 T cells in the spleen than transfer-only mice and these cells also expanded in response to peptide/LPS challenge (Fig. 6C). Although live Salmonella-vaccinated mice had a very low frequency of SM1 T cells in the spleen, these cells expanded after exposure to peptide/LPS. Therefore, the few SM1 T cells remaining in the spleen of mice administered live Salmonella are functionally responsive. The Journal of Immunology In this particular experiment, SM1 T cells had not fully diluted CFSE in response to live Salmonella, allowing clear visualization of divided and undivided SM1 cells. The failure of SM1 T cells to persist at day 10 corresponded with the preferential loss of the most CFSE-dilute cells (Fig. 7A, C57BL/6, day 3 vs day 10). In contrast, SM1 T cells persisted in OT-II recipient mice at day 10, and this included the survival of the most CFSE-dilute cells (Fig. 7A, OT-II, day 3 vs day 10). We conclude that SM1 T cells fail to persist due to competition from the endogenous Salmonella-specific CD4 T cell response to live Salmonella. Flagellin is not a major target Ag of endogenous Salmonella-specific CD4 T cells Discussion We have analyzed the response of CD4 T cells to vaccination with live attenuated S. typhimurium. Our data demonstrate that a large increase in the number of CD4⫹CD11ahigh T cells occurs rapidly after vaccination and is maintained for several weeks. A rise in the number of CD4⫹CD44high cells has also been reported in resistant mice after infection with virulent Salmonella (36). Together these data suggest that a large expansion of CD4 cells with an activated phenotype is typical of the immune response to Salmonella. Our in vivo HKST pulse assay demonstrates that the vast majority of these CD4⫹CD11ahigh cells are Salmonella specific. The 2-h stimulation period of the HKST pulse assay ensures that this assay measures effector cytokine production from previously expanded CD4 cells and not the clonal expansion of CD4 T cells in response to HKST. It is possible that the IFN-␥ production detected using the HKST pulse assay does not derive from Salmonella-specific CD4 T cells. However, we think this is unlikely for the following reasons: First, cytokine production was not detected in the naive CD11alow population and comes solely from cells with an activated CD11ahigh surface phenotype. Therefore, if this assay measures a nonspecific response, it is a nonspecific response that is peculiar to CD4 T cells with an Ag-experienced surface phenotype. Second, injection of HKLM did not allow the detection of CD4⫹IFN-␥⫹ T cells in the spleen. Therefore, if this is a nonspecific response to bacterial inflammation, it is one that appears to be particular to Salmonella Ags. Third, we detected no obvious bias in the CD4 TCR V repertoire of CD4 T cells producing IFN-␥ in the pulse assay, rendering the possibility of a Salmonella SAg in the HKST preparation unlikely. Fourth, there is an expansion of the IFN-␥ response in the first few days after infection and a contraction of this response when bacteria are cleared. Therefore, the kinetics of IFN-␥ production by the CD4⫹CD11ahigh population represent the kinetics of an adaptive immune response rather than an innate response to inflammation. Lastly, a high frequency of CD4⫹CD11a⫹ cells producing IFN-␥ was detected after the complete clearance of attenuated bacteria. Therefore, we can detect immune memory, a hallmark of adaptive immune responses. Taken together, these data support the conclusion that the HKST pulse assay detects Salmonella-specific CD4 T cells in vivo. Although we think the evidence is strong that the assay detects Ag-specific CD4 T cells, we have not conclusively demonstrated that ligation of the TCR is required during the recall response. Therefore, it remains possible that a large number of Salmonella-specific CD4 T cells expand after vaccination but that these cells acquire the ability to respond in an Ag-nonspecific manner to the HKST pulse. Whatever the exact mechanism, the detection of a response months after infection indicates that our assay allows the detection of an adaptive immune response to Salmonella. We have therefore been able to detect a surprisingly large population of Ag-specific CD4 T cells in response to live vaccination. At the peak of the response, this comprises ⬃50% of the entire CD4 population in the spleen and in some experiments can be as high as 65% (data not shown). The detection of an Ag-specific CD4 T cell response of this magnitude is unprecedented. A number of reports have measured the CD4 burst size in response to infection to be ⬃2–3% of all CD4 T cells (12, 16 –19), although one report measured the peak CD4 T cell response to lymphocytic choriomeningitis virus at ⬃10% of CD4 T cells (13). This raises the question of why the Salmonella-specific CD4 response would be so large in response to live vaccination? A simple explanation is that previous studies have examined CD4 T cell responses under conditions where effector CD4 expansion is not absolutely critical for the development of protective immunity. During viral (12, 16 –19) or Listeria infection (21), although CD4 T cells can clearly contribute to the antiviral response (37), the expansion of CD8 effector cells is more likely to be critical for viral clearance. In contrast, CD4 T cells are absolutely required for the clearance of attenuated Salmonella strains in vivo (23, 38). Analysis of the CD4 clonal burst size in other infectious disease models may therefore demonstrate much higher frequencies than those reported for viral infections. However, we favor an alternative hypothesis. We think it possible that the huge expansion of Salmonella-specific CD4 T cells reported here is peculiar to this infectious disease model. This might occur because of the presence of an unusually high frequency of endogenous Salmonella-specific T cells in the secondary lymphoid tissues of naive mice. These cells might arise naturally due to cross-reactivity between Salmonella epitopes and those present in the normal bacterial flora. Indeed, our analysis of naive C57BL/6 mice indicates the presence of a small but visible population of HKST-specific CD4 T cells that produce IFN-␥ (Fig. 3A, naive ⫹ HKST). Future experiments using mice with defined gut flora will help clarify whether these cells arose in response to cross-reactive bacterial epitopes and are responsible for the large clonal burst in response to Salmonella. What Ags are recognized by Salmonella-specific CD4 T cells in vivo? Only two Ags have been clearly defined as targets of Salmonella-specific T cells, flagellin and SipC (29, 35, 39). Our experiments suggest that flagellin is not a major target of CD4 T cells in vivo, despite its identification as a target of numerous Salmonella-specific T cell clones in vitro (29, 35). It is possible that the intrinsic adjuvant properties of flagellin (40), mediated through Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 It still was not clear why endogenous CD4 T cells would have a selective survival advantage over SM1 T cells. In fact, previous adoptive transfer experiments have reported that an increased frequency of TCR-transgenic cells actually blocks the clonal expansion of endogenous T cells (3, 6, 34). Although flagellin has been identified as a target of Salmonella-specific CD4 T cell clones (29, 35), it remained possible the majority of the endogenous CD4 response was directed at other unknown proteins and that flagellinspecific CD4 T cells do not compete well with these other specificities. We therefore sought to use the HKST pulse assay to estimate the fraction of endogenous Salmonella-specific CD4 T cells that are specific for flagellin after live vaccination. HKST was prepared from flagellate or aflagellate Salmonella and these Ag preparations compared for their ability to activate endogenous CD4 T cells in vaccinated mice. Surprisingly, we found that the endogenous CD4 response to flagellate HKST was roughly equivalent to that of aflagellate HKST (Fig. 7B), suggesting that flagellin is not a major target of Salmonella-specific CD4 T cells in vivo. Therefore, it is likely that SM1 T cells do not compete well with the endogenous Salmonella-specific response due to differences in the Ag specificity of these two populations. 6891 6892 Acknowledgments We thank Dr. A. Vella and Dr. L. Lefrancois for helpful discussion. References 1. Burnet, F. M. 1957. The clonal selection theory. Aust. J. Sci. 20:67. 2. Casrouge, A., E. Beaudoing, S. Dalle, C. Pannetier, J. Kanellopoulos, and P. Kourilsky. 2000. Size estimate of the ␣ TCR repertoire of naive mouse splenocytes. J. Immunol. 164:5782. 3. Blattman, J. N., R. Anita, D. J. D. Sourdive, X. Wang, S. M. Kaech, K. Murali-Krishna, J. D. Altman, and R. Ahmed. 2002. Estimating the precursor frequency of naive antigen-specific CD8 T cells. J. Exp. Med. 195:657. 4. Ahmed, R., and D. Gray. 1996. Immunological memory and protective immunity: understanding their relation. Science 272:54. 5. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. D. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177. 6. Butz, E. A., and M. J. Bevan. 1998. Massive expansion of antigen-specific CD8⫹ T cells during an acute virus infection. Immunity 8:167. 7. Kedl, R. M., W. A. Rees, D. A. Hildeman, B. Schaefer, T. Mitchell, J. Kappler, and P. Marrack. 2000. T cells compete for access to antigen-bearing antigenpresenting cells. J. Exp. Med. 192:1105. 8. Chen, W., L. C. Anton, J. R. Bennink, and J. W. Yewdell. 2000. Dissecting the multifactorial causes of immunodominance in class I-restricted T cell responses to viruses. Immunity 12:83. 9. Kedl, R. M., J. W. Kappler, and P. Marrack. 2003. Epitope dominance, competition and T cell affinity maturation. Curr. Opin. Immunol. 15:120. 10. Probst, H. C., T. Dumrese, and M. F. van den Broek. 2002. Cutting edge: competition for APC by CTLs of different specificities is not functionally important during induction of antiviral responses. J. Immunol. 168:5387. 11. Seder, R. A., and R. Ahmed. 2003. Similarities and differences in CD4⫹ and CD8⫹ effector and memory T cell generation. Nat. Immunol. 4:835. 12. Whitmire, J. K., K. Murali-Krishna, J. Altman, and R. Ahmed. 2000. Antiviral CD4 and CD8 T-cell memory: differences in the size of the response and activation requirements. Philos. Trans. R. Soc. London B 355:373. 13. Homann, D., L. Teyton, and M. B. Oldstone. 2001. Differential regulation of antiviral T-cell immunity results in stable CD8⫹ but declining CD4⫹ T-cell memory. Nat. Med. 7:913. 14. Harrington, L. E., R. Most Rv, J. L. Whitton, and R. Ahmed. 2002. Recombinant vaccinia virus-induced T-cell immunity: quantitation of the response to the virus vector and the foreign epitope. J. Virol. 76:3329. 15. Cauley, L. S., T. Cookenham, T. B. Miller, P. S. Adams, K. M. Vignali, D. A. Vignali, and D. L. Woodland. 2002. Cutting edge: virus-specific CD4⫹ memory T cells in nonlymphoid tissues express a highly activated phenotype. J. Immunol. 169:6655. 16. Whitmire, J. K., M. S. Asano, K. Murali-Krishna, M. Suresh, and R. Ahmed. 1998. Long-term CD4 Th1 and Th2 memory following acute lymphocytic choriomeningitis virus infection. J. Virol. 72:8281. 17. Topham, D. J., and P. C. Doherty. 1998. Longitudinal analysis of the acute Sendai virus-specific CD4⫹ T cell response and memory. J. Immunol. 161:4530. 18. Christensen, J. P., and P. C. Doherty. 1999. Quantitative analysis of the acute and long-term CD4⫹ T-cell response to a persistent gammaherpesvirus. J. Virol. 73:4279. 19. Kamperschroer, C., and D. G. Quinn. 1999. Quantification of epitope-specific MHC class-II-restricted T cells following lymphocytic choriomeningitis virus infection. Cell. Immunol. 193:134. 20. Varga, S. M., and R. M. Welsh. 1998. Detection of a high frequency of virusspecific CD4⫹ T cells during acute infection with lymphocytic choriomeningitis virus. J. Immunol. 161:3215. 21. Foulds, K. E., L. A. Zenewicz, D. J. Shedlock, J. Jiang, A. E. Troy, and H. Shen. 2002. Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses. J. Immunol. 168:1528. 22. Kaufmann, S. H. 1993. Immunity to intracellular bacteria. Annu. Rev. Immunol. 11:129. 23. 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-␣ cells and IFN-␥ in bacterial clearance independent of intracellular location. J. Immunol. 156:3321. 24. Nauciel, C. 1990. Role of CD4⫹ T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection. J. Immunol. 145:1265. 25. McSorley, S. J., S. Asch, M. Costalonga, R. L. Rieinhardt, and M. K. Jenkins. 2002. Tracking Salmonella-specific CD4 T cells in vivo reveals a local mucosal response to a disseminated infection. Immunity 16:365. 26. Barnden, M. J., J. Allison, W. R. Heath, and F. R. Carbone. 1998. Defective TCR expression in transgenic mice constructed using cDNA-based ␣- and -chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76:34. 27. Parish, C. R. 1999. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77:499. 28. Strugnell, R., G. Dougan, S. Chatfield, I. Charles, N. Fairweather, J. Tite, J. L. Li, J. Beesley, and M. Roberts. 1992. Characterization of a Salmonella typhimurium aro vaccine strain expressing the P.69 antigen of Bordetella pertussis. Infect. Immun. 60:3994. 29. McSorley, S. J., B. T. Cookson, and M. K. Jenkins. 2000. Characterization of CD4⫹ T cell responses during natural infection with Salmonella typhimurium. J. Immunol. 164:986. 30. Khoruts, A., A. Mondino, K. A. Pape, S. L. Reiner, and M. K. Jenkins. 1998. A natural immunological adjuvant enhances T cell clonal expansion through a CD28-dependent, interleukin (IL)-2-independent mechanism. J. Exp. Med. 187:225. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 TLR-5 (41, 42), favor the outgrowth of flagellin-specific CD4 T cells in vitro. However, this conclusion may be premature, and future studies using recombinant Salmonella Ags in the in vivo pulse assay will define the frequency of flagellin-specific CD4 T cells in vivo. Identification of the Ags and epitopes recognized by endogenous Salmonella-specific T cells in vivo will increase our understanding of the CD4 response to Salmonella, help drive the generation Ag-specific detection reagents in this particular model, and aid the rational development of vaccines against typhoid fever. Although we are unable to determine the antigenic targets of this response in vivo, our in vivo pulse assay appears to be a simple means to detect pathogen-specific T cells in vivo and should therefore be adaptable to other infectious disease models where Agspecific reagents are also scarce. The peak endogenous CD4 response to Salmonella appears to be sustained for several weeks after infection, yet most in vivo studies on T cells have reported rapid progression from the expansion to contraction phase. However, it has been demonstrated that Agspecific T cells preferentially persist in nonlymphoid tissues where Ag is present (43). We think it likely that the maintenance of the response in the spleen reflects a similar retention of expanded Salmonella-specific T cells due to infection of this particular lymphoid tissue. Why does the expansion of endogenous Salmonella-specific CD4 cells hinder the persistence of SM1 T cells? It is particularly interesting that SM1 T cells fail to persist despite their artificially enhanced frequency in the naive T cell pool and their extensive proliferation in response to live vaccination. Usually these factors would favor the survival of adoptively transferred T cells at the expense of the endogenous population (3, 6, 34). It is therefore noteworthy that SM1 T cells only fail to persist in response to live vaccination and that clonal expansion of the endogenous response appears to be greatest under these conditions. It is likely that under conditions of greatest clonal expansion there would be greatest competition for resources, such as access to APC or an essential growth factor. Such competition would be less intense during immunization with HKST or peptide/LPS, as clonal expansion of endogenous T cells is lower. The persistence of SM1 T cells after vaccination of adoptively transferred OT-II mice suggests strongly that the endogenous CD4 response to Salmonella inhibits the persistence of SM1 T cells. The mechanism of such inhibition is not clear and may reflect competition for survival signals directly after clonal expansion. It is unclear why SM1 T cells would be at a disadvantage in such direct competition. It is unfortunate that our system to detect competition also results in the homeostatic expansion of SM1 T cells in vivo. Although we think it unlikely that homeostatic expansion accounts for the enhanced persistence of SM1 T cells in OT-II mice following live vaccination, we cannot rule out this possibility. Therefore, it remains possible that other noncompetitive mechanisms contribute to the decay of SM1 T cells in live vaccinated mice. In conclusion, our data demonstrate that a massive response of Salmonella-specific CD4 T cells and interclonal competition occur after vaccination with live Salmonella, but not after immunization with HKST. These data may explain the success of live attenuated Salmonella vaccines in vivo (44) and should help the rational design of more effective prophylactic measures against typhoid fever in the future. CD4 T CELL RESPONSE TO LIVE ATTENUATED Salmonella The Journal of Immunology 31. Reinhardt, R. L., A. Khoruts, R. Merica, T. Zell, and M. K. Jenkins. 2001. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410:101. 32. Li, H., A. Llera, E. L. Malchiodi, and R. A. Mariuzza. 1999. The structural basis of T cell activation by superantigens. Annu. Rev. Immunol. 17:435. 33. Wu, C. Y., J. R. Kirman, M. J. Rotte, D. F. Davey, S. P. Perfetto, E. G. Rhee, B. L. Freidag, B. J. Hill, D. C. Douek, and R. A. Seder. 2002. Distinct lineages of TH1 cells have differential capacities for memory cell generation in vivo. Nat. Immunol. 3:852. 34. Smith, A. L., M. E. Wikstrom, and B. Fazekas de St Groth. 2000. Visualizing T cell competition for peptide/MHC complexes: a specific mechanism to minimize the effect of precursor frequency. Immunity 13:783. 35. Cookson, B. T., and M. J. Bevan. 1997. Identification of a natural T cell epitope presented by Salmonella-infected macrophages and recognized by T cells from orally immunized mice. J. Immunol. 158:4310. 36. Mittrucker, H., A. Kohler, and S. H. Kaufmann. 2002. Characterization of the murine T-lymphocyte response to Salmonella enterica serovar typhimurium infection. Infect. Immun. 70:199. 37. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. D. Sourdive, M. Suresh, J. D. Altman, and R. Ahmed. 1998. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188:2205. 6893 38. Sinha, K., P. Mastroeni, J. Harrison, R. D. de Hormaeche, and C. E. Hormaeche. 1997. Salmonella typhimurium aroA, htrA, and AroD htrA mutants cause progressive infections in athymic (nu/nu) BALB/c mice. Infect. Immun. 65:1566. 39. Musson, J. A., R. D. Hayward, A. A. Delvig, C. E. Hormaeche, V. Koronakis, and J. H. Robinson. 2002. Processing of viable Salmonella typhimurium for presentation of a CD4 T cell epitope from the Salmonella invasion protein C (SipC). Eur. J. Immunol. 32:2664. 40. McSorley, S. J., B. D. Ehst, Y. Yu, and A. T. Gewirtz. 2002. Bacterial flagellin is an effective adjuvant for CD4 T cells in vivo. J. Immunol. 169:3914. 41. Gewirtz, A. T., T. A. Nava, S. Lyons, P. J. Godowski, and J. L. Madera. 2001. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J. Immunol. 167:1882. 42. Hayashi, F., K. D. Smith, A. Ozinsky, T. R. Hawn, E. C. Yi, D. R. Goodlett, J. K. Eng, S. Akira, D. M. Underhill, and A. Aderem. 2001. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410:1099. 43. Reinhardt, R. L., D. C. Bullard, C. T. Weaver, and M. K. Jenkins. 2003. Preferential accumulation of antigen-specific effector CD4 T cells at an antigen injection site involves CD62E-dependent migration but not local proliferation. J. Exp. Med. 197:751. 44. Hoiseth, S. K., and B. A. D. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291:238. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017