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* Department of Microbiology and
Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA 52242
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD8+ T cell response to LM infection has been studied extensively (14). The peak in the number of Ag-specific CD8+ T cells in the spleen occurs between 7 and 9 days postinfection (15, 16, 17, 18) and is followed by a rapid contraction (
90%) to memory cell numbers that can remain stable for the life of the host. Although there is much information relating to how naive and memory T cells are activated and maintained, the mechanism regulating the transition between expansion and contraction of Ag-specific T cells is largely unknown. In many acute infection systems, the onset of the T cell contraction phase correlates with pathogen clearance (19), suggesting that the duration of infection determines when Ag-specific CD8+ T cells transition between the expansion and contraction phases of the immune response. However, recent data revealed that ampicillin treatment early after infection resulted in earlier clearance of LM infection and shortened duration of Ag display, but did not shorten the interval between initial infection and the onset of the CD8+ T cell contraction phase (17). Similarly, the onset of CD8+ T cell contraction is largely unaffected in mice with prolonged Ag display as a result of chronic lymphocytic choriomeningitis virus infection (17, 20, 21). Thus, the durations of infection and Ag display do not determine the onset of Ag-specific CD8+ T cell contraction, suggesting that the contraction phase is programmed by early events after infection.
In contrast, much less is known about the CD4+ T cell response to LM infection. CD4+ T cells contribute to immunity by promoting dendritic cell (DC) activation (22), secreting inflammatory cytokines that activate the antimicrobial activities of macrophages, and providing help for the Ab response (23). In general, activation of both CD4+ and CD8+ T cell responses requires Ag and costimulation (24, 25), and both cell populations exhibit similar overall kinetics of expansion and contraction to memory (26). However, comparison of CD4+ and CD8+ T cell responses to infection have revealed differences that may reflect their differing roles in immunity. For instance, the kinetics of CD4+ and CD8+ T cell responses are synchronized, but over the life of the host, stable numbers of Ag-specific memory CD8+ T cells persist, whereas CD4+ T cell memory declines over time (26, 27). In addition, CD8+ T cell memory, but not CD4+ T cell memory, is reduced after heterologous infection (28). Differences in CD4+ and CD8+ T cell memory maintenance may reflect different requirements for cytokines or other signals involved in the survival and proliferation of these cells. Reports show that IL-7 promotes memory CD4+ and CD8+ T cell survival (29, 30, 31, 32), whereas IL-15 regulates memory CD8+, but not memory CD4+, T cell proliferation (33, 34, 35, 36, 37). Thus, there are both similarities and differences between the CD8+ and CD4+ T cell responses to infection.
It is currently unknown whether the kinetics of the CD4+ T cell response are dictated by the duration of infection and Ag display or are programmed early after infection, as seen with the CD8+ T cell response. To address this issue, we compared Ag-specific CD4+ and CD8+ T cell responses in C57BL/6 mice after infection with LM in control mice or those treated with antibiotic to truncate infection. We also examined the resulting memory T cells for the ability to carry out secondary CD4+ and CD8+ T cell responses and provide immunity to LM challenge.
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Materials and Methods |
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C57BL/6 (B6) mice were obtained from the National Cancer Institute (Frederick, MD). B6.PL-Thy-1a (B6.PL) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained by brother-sister mating under specific pathogen-free conditions. Mice were initially used at 6–10 wk of age.
Bacteria and infection of mice
Recombinant LM expressing secreted OVA protein (rLM-OVA) (38) was provided by H. Shen (University of Pennsylvania School of Medicine, Philadelphia, PA). Bacteria were grown in tryptic soy broth with 50 µg/ml streptomycin to OD600 0.1, then diluted in 0.9% sodium chloride (Abbott Laboratories, Chicago, IL), and 104 CFU of rLM-OVA was injected i.v. as previously described (39). The actual number of bacteria injected was confirmed by plate count. Some mice received ampicillin (2 mg/ml; Sigma-Aldrich, St. Louis, MO) in their drinking water beginning at various times postinfection; these mice were maintained on oral ampicillin for 7 days. In some experiments, mice were rechallenged with 5 x 105 CFU of rLM-OVA on the indicated days after primary infection. CFU per spleen were determined on various days after infection as previously described (40).
CD4+ T cell line
A CD4+ T cell line specific for LLO190–201 (41) was derived from B6 mice injected 30 days previously with 1 x 104 CFU of rLM-OVA. Briefly, 4 x 107 splenocytes from these mice were incubated in RP10 (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 µg/ml gentamicin, 10 mM HEPES, 2 mM L-glutamine, and 50 µM 2-ME) at 37°C in 7% CO2 with 1.6 µg/ml (1 µM) LLO190–201 peptide. Subsequent restimulations were performed every 2 wk by combining 3 x 106 T cells with 5 x 107 irradiated (30 Gy) syngenic splenocytes that had been coated with 1.6 µg/ml LLO190–201 peptide for 30 min, followed by two washes in RP10. T cells and peptide-coated splenocytes were incubated in RP10 supplemented with 5% supernatant from Con A-stimulated rat spleen cells and 50 mM 
-methyl mannoside (42).
Abs, peptides, and detection of Ag-specific T cells
The following mAbs were obtained from BD Pharmingen (San Diego, CA): PE-anti-TNF-, PE-anti-IFN-
, PerCP-anti-CD4, PerCP-anti-CD8, FITC-anti-CD90.1, and allophycocyanin-anti-CD62L. Synthetic peptides LLO190–201 (NEKYAQAYPNVS) and OVA257–264 (SIINFEKL) were purchased from Biosynthesis International (Lewisville, TX). The magnitude of epitope-specific CD4+ and CD8+ T cell responses were determined by intracellular cytokine staining (ICS) for IFN- using a Cytofix/Cytoperm Plus (with GolgiPlug) Kit (BD Pharmingen) as previously described (43). Splenocytes from each infected mouse were treated with ACK lysis buffer for 5 min at room temperature to remove RBC. Splenocytes were washed twice in RP10 and resuspended in the same medium in 5 ml. Cells (200 µl) were incubated for 6 h at 37°C with medium alone or with LLO190–201 peptide (5 µM) or OVA257–264 peptide (100 nM) in the presence of 1 µl/ml GolgiPlug (brefeldin A). Cells were washed in FACS buffer (PBS supplemented with 1% FCS and 200 mg/ml NaN3) and were incubated with anti-FcII/III (2.4G2) and CyChrome-labeled anti-CD4 or anti-CD8 Ab at 4°C for 30 min. The cells were washed in FACS buffer, then fixed and permeabilized by incubation for 15 min in 250 µl of Cytofix/Cytoperm solution. Then cells were washed in Perm/wash solution and stained with PE-anti-IFN- or PE-anti-TNF- for 30 min at 4°C. Cells were washed twice in Perm/wash solution and resuspended in 300 µl of FACS buffer before flow cytometric analysis. List mode data were acquired on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) using CellQuest software. Data was analyzed with FlowJo software (Tree Star, San Carlos, CA). The gate for IFN-+ cells was selected such that the percentage of IFN-+ cells in the unstimulated sample for each mouse was <0.5 ± 0.1% of the CD4+ or CD8+ splenocytes. The percentage of Ag-specific T cells was calculated by subtracting the peptide-unstimulated value from the peptide-stimulated value. The total number of epitope-specific CD4+ or CD8+ T cells per spleen was calculated from the percentage of IFN-+CD4+ or CD8+ T cells, the percentage of CD4+ or CD8+ cells in each sample, and the total number of cells per spleen.
Direct ex vivo Ag detection (DEAD) assay
The DEAD assay was preformed as previously described (17), except that an LLO190–201-specific CD4+ T cell line was used to measure LLO190–201 Ag display. Briefly, 5 x 104 CFSE (Molecular Probes, Eugene, OR)-labeled, LLO190–201-specific CD4+ T cells were mixed with 1 x 106 splenocytes obtained at various times from rLM-OVA-infected mice. Cultures were incubated for 12 h, with brefeldin A (Sigma-Aldrich) added for the last 6 h of incubation. CFSE-labeled cells were then analyzed by ICS for IFN- or TNF. LLO190–201-specific CD4+ T cells incubated with naive splenocytes in the presence or the absence of synthetic LLO190–201 peptide served as positive and negative controls, respectively. Splenocytes prepared with or without DNase/collagenase treatment (44) were comprised of a similar fraction of CD11c+ cells (2.5–3%) and had a similar level of Ag, as detected by the DEAD assay (data not shown).
DC depletion
CD11c+ cells were depleted from splenocyte preparations by incubation with N418-FITC for 15 min at 4°C. After incubation, cells were washed and resuspended with 100 µl of mircobeads (coated with anti-FITC Ab; Miltenyi Biotech, Auburn, CA) and 900 µl of PBS for 15 min at 4°C. Cells were passed over an LS MACS separation column (Miltenyi Biotech). Flow-through was collected, washed twice in RP10, and resuspended at 1 x 106 cells/ml. This depletion procedure was effective in reducing the percentage of CD11c to <0.8% of the total splenocytes.
ELISPOT-based Ag detection assay
Ag presentation by in vivo-infected splenocytes was also assessed with an ELISPOT-based assay as previously described (45), except that Ag was detected using CD4-enriched splenocytes from rLM-OVA memory mice. Splenocyte cell suspensions were enriched for CD4+ T cells by negative selection (StemCell Technologies, Vancouver, Canada). Briefly 8 x 107 splenic leukocytes/ml were labeled with a CD4+ enrichment Ab mixture, followed by conjugation to magnetic beads. CD4+ T cells were obtained in the flow-through after magnetic separation (Macs LS separation column; Miltenyi Biotec). Recovered cells were >95% CD4+ by flow cytometry.
Graded numbers of splenocytes obtained from mice between 1 and 3 days after primary rLM-OVA infection were cultured with 3 x 105 CD4-enriched splenocytes from LM memory mice in round-bottom, 96-well microtiter plates in a final volume of 100 µl of RP10. Plates were incubated for 5 h at 37°C 7% CO2. After preincubation, cells were resuspended and transferred to rat anti-mouse IFN- mAb-coated (1/100 dilution of RMMG-1 (BioSource International, Camarillo, CA) in PBS), nylon membrane-backed, 96-well microtiter plates (Nunc, Wiesbaden, Germany) and incubated overnight. After lysis of cells with distilled water and rinses with PBS with 0.05% Tween 20 (PBS-T), ELISPOT plates were incubated overnight at 4°C with a 1/1000 dilution of rabbit anti-IFN- antiserum (gift from Dr. J. Cowdery, University of Iowa, Iowa City, IA) in PBS-T. After washing three times in PBS-T, donkey anti-rabbit Ig conjugated to alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) was added according to the manufacturer’s instructions. After overnight incubation at 4°C, plates were developed with the Alkaline Phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. The reaction was developed for 30 min at room temperature, and spots were counted using a dissecting microscope. The number of spots observed in wells containing infected splenocytes alone was subtracted from the number of spots in test wells (CD4-enriched memory splenocytes plus infected splenocytes).
Adoptive transfer experiments
B6.PL (Thy1.1) mice were infected with rLM-OVA. Seventy days after infection, 3 x 106 splenocytes were analyzed for CD4+ LLO190–201-specific T cells and CD8+ OVA257–264-specific T cells to determine the number of Ag-specific memory T cells present. Splenocytes were transferred i.v. into naive B6 (Thy1.2) mice to generate recipients containing the indicated number of Ag-specific memory cells (Thy1.1) in addition to endogenous naive precursors (Thy1.2). Recipient mice were infected 1 day later with 1 x 104 CFU of rLM-OVA. On the indicated days after challenge, recipient mice were euthanized, and spleens were taken for analysis.
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Results |
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We examined whether the kinetics of the CD4+ T cell response to LM infection were similarly programmed by early events after infection, as shown for the CD8+ T cell response of BALB/c mice (17). However, no strong CD4+ T cell epitopes are defined from LM-infected BALB/c mice. Thus, we could not use the BALB/c model to study concurrent CD4+ T cell and CD8+ T cell responses to infection. In contrast, LM infection of B6 mice stimulates a very strong, I-Ab-restricted, CD4+ T cell response to aa 190–201 of the LLO protein (LLO190–201) (41). However, no strong MHC class Ia-restricted CD8+ T cell epitopes are identified in LM-infected B6 mice. To circumvent this problem, we infected B6 mice with a recently described recombinant LM strain expressing hen OVA (rLM-OVA) as a secreted protein (38). Infection of B6 mice with rLM-OVA stimulates a vigorous expansion and the generation of memory CD4+ T cells specific for LLO190–201 as well as a strong H-2Kb-restricted CD8+ T cell response to the well-characterized OVA257–264 epitope (26). This system allowed us to directly compare CD4+ T cells and CD8+ T cell responses in the same host.
To investigate the role of duration of infection in both CD4+ and CD8+ T cell responses, rLM-OVA infection was attenuated in some mice with antibiotics, as previously described for analysis of the CD8+ T cell response in BALB/c mice (17). Administration of ampicillin to mice via the drinking water beginning at 24 h postinfection resulted in an 20-fold reduction in bacterial numbers 1 day later and complete clearance of bacteria by day 3 compared with clearance by day 7 in control infected mice (Fig. 1A). Thus, ampicillin treatment at 24 h postinfection results in rapid clearance of bacteria from infected mice and truncates the infection by 4 days compared with control infected mice.
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, as detected by ICS, is dependent on the dose of Ag (Fig. 1B). On day 2 postinfection, a substantially lower amount of the LLO190–201 Ag display was detected in spleen cells prepared from representative mice that had received ampicillin treatment 24 h postinfection, compared with the amount in mice that did not receive ampicillin (Fig. 1C). A kinetic analysis revealed that whereas LLO190–201 Ag display in control mice slowly declines between days 1–7 postinfection, ampicillin treatment at 24 h caused a rapid reduction in the amount of LLO190–201 that fell below the level of detection on day 5, at least 2 days earlier than in control mice (Fig. 1D). Similar results were obtained using TNF production by the LLO190–201-specific CD4+ T cell line as the readout (data not shown). The DEAD assay we used detects Ag display by any infected cell. However, it is likely that DC Ag display is the most meaningful in terms of stimulating naive T cell responses. To assess the contribution of DCs to MHC class II LLO190–201 display in the spleen, we depleted CD11c+ DCs by negative selection. We found that MHC class II LLO190–201 display on day 1 postinfection was significantly decreased after depletion of CD11c+ DC (Fig. 1E), suggesting that DCs account for at least some of the LLO190–201 Ag detectable in the spleen using the DEAD assay.
The LLO190–201-specific CD4+ T cell line used to probe LLO190–201 Ag display in the DEAD assay exhibits detectable background IFN- production (Fig. 1C; 5% IFN-+CD4+ T cells) in the presence of naive splenocytes. To confirm the results of the DEAD assay, we performed an ELISPOT-based Ag detection assay (45) using LLO190–201-specific memory CD4+ T cells, in the form of CD4-enriched splenocytes from rLM-OVA immune mice, to probe Ag display on splenocytes from infected mice. The maximal number of spots (150/well) was detected at high input peptide amounts and decreased as the dose of peptide was reduced (Fig. 1F). Thus, the number of spots in the ELISPOT assay is saturable, but still can be used as a relative measure of Ag display. Similarly, the number of spots decreased as the input number of spleen cells from day 1 infected mice was reduced (Fig. 1G). On day 3, only a few spots were detected after culture of memory CD4+ T cells, with the highest input number of spleen cells from ampicillin-treated mice, whereas the same number of spleen cells from control mice stimulated a saturating response (150 spots). Thus, the results of the ELISPOT-based detection assay were in agreement with the results obtained using the DEAD assay and showed that ampicillin treatment reduces the amount of LLO190–201 Ag display in the spleen early after infection. Together, these data suggest a direct relationship between the number of bacteria and the amount of LLO190–201 Ag display after rLM-OVA infection.
CD4+ and CD8+ T cell responses after infection and antibiotic treatment
To determine the effect of shortening the duration of infection and Ag display on the kinetics of the CD4+ and CD8+ T cell response in the same host, we measured LLO190–201-specific CD4+ and OVA257–264-specific CD8+ T cell responses on various days after rLM-OVA infection in control mice and in mice that received ampicillin treatment at 24 h postinfection (Fig. 2). The LLO190–201-specific CD4+ T cell response in mice infected with rLM-OVA and treated with ampicillin 24 h after infection was robust on day 7 postinfection and similar to the response in controls that did not receive ampicillin (Fig. 2, A and B). The peak of the LLO190–201-specific CD4+ T cell response was observed on day 8 in control mice and on day 7 in ampicillin-treated mice. However, the expansion in number between days 7 and 8 in control mice was <2-fold. Despite the apparent shift in the peak of the response, there was no significant difference in the rate or magnitude of contraction when the CD4+ T cell responses in ampicillin-treated and control mice were normalized to the respective peak days for each group (Fig. 2C). The numbers of LLO190–201-specific CD4+ T cells that remained after the contraction phase on day 28 were also not statistically different in ampicillin-treated and control mice. In general, ampicillin treatment had the same effect on the OVA257–264-specific CD8+ T cell response in the same mice (Fig. 2, D–F). These data show that ampicillin treatment, which substantially shortened the duration of infection and Ag display, had a minimal impact on the kinetics of the CD4+ T cell response after LM infection. Thus, the kinetics of the CD4+ and CD8+ T cell response in B6 mice are also largely independent of prolonged infection and Ag display.
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To determine the interval of infection needed for a full CD4+ and CD8+ T cell response on day 7, mice were inoculated with rLM-OVA and treated with ampicillin at 0, 24, or 48 h after infection. Bacterial clearance below the limit of detection (50 CFU/spleen) was observed at 1–2 days after ampicillin treatment for all groups (Fig. 3A). In mice that received ampicillin at 0, 24, or 48 h postinfection, the peak of the CD4+ and CD8+ T cell responses was observed on day 7 compared with control mice, in which the peak of the T cell responses was observed on day 8 postinfection (data not shown). In mice that received ampicillin immediately after infection, the LLO190–201-specific CD4+ T cell response on day 7 was 10-fold lower than the control response (Fig. 3B). Delaying ampicillin until 24 h after infection resulted in an LLO190–201-specific T cell response that was significantly lower than the control response, albeit the reduction was only 2-fold. Similar levels of LLO190–201-specific T cells relative to control were observed if ampicillin treatment was delayed until 48 h after infection. These data show that CD4+ T cells require 48 h of unmodified infection to generate the full response on day 7. As for the OVA257–264-specific CD8+ T cell response, a slight reduction on day 7 was seen in mice given ampicillin immediately after infection, compared with control animals (Fig. 3D). Mice that received ampicillin at 0 or 24 h after infection had OVA257–264-specific CD8+ T cell responses on day 7 that were similar to those in controls. These data suggest that CD8+ T cells only require 24 h of unmodified infection to generate the full response on day 7. Together, the results are consistent with the idea that a longer window of unmodified bacterial infection is required to activate or recruit the full complement of naive Ag-specific CD4+ T cells compared with CD8+ T cells after LM infection. Although the timing of antibiotic treatment did affect the magnitude of the response, it did not alter the overall kinetics of the CD4+ or CD8+ T cell response, because the expansion and contraction phases occurred with similar kinetics in all groups (Fig. 3, C and E).
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The data presented in this study show that ampicillin treatment after immunization with rLM-OVA has, at most, a modest impact on the number of CD4+ or CD8+ memory T cells on day 28 postinfection. However, the functionality of these cells may be altered by ampicillin treatment shortly after infection, as previous studies suggested that abridgement of LM infection with antibiotics resulted in decreased protection against a subsequent challenge (46). To determine whether ampicillin treatment after primary inoculation impacts the secondary T cell response, we challenged control immune or ampicillin-treated immune mice on day 28 postprimary infection with a high dose of rLM-OVA and examined the secondary Ag-specific CD4+ and CD8+ T cell responses. There was no difference in the kinetics or magnitude of secondary LLO190–201-specific CD4+ T cell expansion in ampicillin-treated immune and control immune mice (Fig. 4A). Similarly, there was no difference in the kinetics or magnitude of secondary OVA257–264-specific CD8+ T cell expansion in the same mice (Fig. 4B). These results show that ampicillin treatment after primary rLM-OVA infection does not alter secondary expansion of memory CD4+ or CD8+ T cells in response to secondary rLM-OVA infection. Comparing CD4+ and CD8+ secondary T cell responses, greater expansion from days 0–5 after rechallenge was observed for OVA257–264-specific CD8+ T cells (300-fold increase) than for LLO190–201-specific CD4+ T cells (60-fold increase; Fig. 4C). Nevertheless, both the CD4+ and CD8+ T cell secondary responses expanded faster and reached a higher number than that seen in the primary responses. Consistent with the high level of secondary T cell expansion in ampicillin-treated immune mice after rLM-OVA rechallenge, the level of bacteria recovered from the spleens of these mice was dramatically lower than that in LM-infected naive animals (Fig. 4C). On day 3 after rechallenge ampicillin-treated immune mice had a slightly higher bacterial load than control immune mice, which may be a consequence of the slightly reduced number of OVA257–264 CD8+ T cells. However, the reduction in bacterial numbers in ampicillin-treated immune mice compared with nonimmune mice was >10,000-fold, and both the ampicillin-treated immune mice and the control immune mice survived the infection and had no detectable bacteria in the spleen on day 5. In contrast, three of four naive mice challenged with rLM-OVA in this experiment died by day 5 postchallenge. Thus, we conclude that substantial protective immunity to lethal rLM-OVA rechallenge developed in B6 mice that received ampicillin treatment at 24 h after primary infection.
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We and others previously showed that contraction of the secondary CD8+ T cell response is prolonged compared with contraction of the primary CD8+ T cell response, independent of host environment or duration of infection (17, 47, 48). To investigate the kinetics of primary vs secondary CD4+ T cell responses and compare the kinetics of CD4+ and CD8+ T cell responses, day 70 memory T cells from rLM-OVA-immune B6.PL mice (Thy1.1+) were transferred into naive B6 recipients (Thy1.2+), and on 1 day post-transfer, recipient mice were infected with rLM-OVA. After infection, the endogenous primary response and secondary response of transferred LLO190–201-specific CD4+ T cells and OVA257–264-specific CD8+ T cells were measured by ICS (Fig. 5B). The endogenous primary CD4+ T cell response showed normal kinetics of expansion and contraction, such that 10% of the cells present at the peak of the expansion remained on day 13 postinfection (Fig. 5C). The secondary response of the transferred memory LLO190–201-specific CD4+ T cells displayed delayed contraction compared with the primary response in the same animals, such that 25% of the cells responding for the second time to infection were present on day 13. Consistent with previous results (17, 48), the contraction of the secondary CD8+ T cell response was also delayed compared with the primary response in the same host (Fig. 5D). These data show that contraction of both CD4+ and CD8+ secondary T cell responses are delayed compared with primary CD4+ and CD8+ T cell responses in the same host.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The factors that determine the timing and magnitude of the contraction phase are unknown. The timing of contraction suggests that T cells sense the clearance of infection, then initiate the contraction phase, as survival cytokines become limiting (19, 49, 50, 51). This model is consistent with the suggestion that a long period of Ag exposure, due to protracted clearance of infection, results in "fit" T cells that proliferate extensively and differentiate to long-lived memory T cells, whereas a short exposure to Ag results in abortive T cell proliferation, and tolerance (52, 53, 54, 55, 56, 57). Both models predict that the duration of infection will determine the timing of the transition between contraction of Ag-specific T cells and the generation of memory. In contrast to this idea, we previously showed in BALB/c mice that ampicillin treatment after infection with attenuated LM does not alter the kinetics of the CD8+ T cell response (17). This suggests that once the CD8+ T cell response is initiated, it carries out the full program of expansion, contraction, and memory. In this study we used ampicillin treatment after LM infection of B6 mice to truncate the duration of infection and Ag display and determined the impact on Ag-specific CD4+ and CD8+ T cell responses and the generation of memory. Our results suggest that, similar to the CD8+ T cell response, early events after infection also program the major features of the CD4+ T cell response to infection.
We previously showed in BALB/c mice that ampicillin treatment at 24 h after infection with attenuated LM did not result in an earlier onset of contraction (17). In the current study we saw a slight reduction (2-fold) in the magnitude of expansion and a shift in the peak of both the CD4+ and CD8+ T cell responses to 1 day earlier in ampicillin-treated mice compared with control infected mice. This may reflect the use of a virulent LM strain in the current study, because the peak CD8+ T cell response occurs 1–2 days later than that after attenuated LM infection (17). Alternatively, the difference may be due to the use of different mouse strains (B6 vs BALB/c) or the different T cell Ag specificities analyzed in the two studies. However, consistent with previous studies (17), we found that shortening LM infection with antibiotic treatment has only a minimal impact on the kinetics of CD4+ and CD8+ T cell responses.
In previous studies it was shown that only 4–8 h of in vitro Ag exposure was required to stimulate multiple rounds of proliferation by transgenic CD8+ T cells (58), whereas 20 h of Ag exposure was required to stimulate proliferation of transgenic CD4+ T cells (52, 59). Together, these results suggest that CD4+ T cells may require more time than CD8+ T cells for activation in response to Ag. Consistent with this idea, we show that a longer interval of unmodified infection is needed for full expansion on day 7 of the CD4+ T cell response compared with the CD8+ T cell response in the same host. The in vivo setting is more complex than the in vitro setting, because differences may exist in the kinetics of MHC class II and class I peptide/MHC complex generation or in the kinetics of APC encounter with T cells. However, we showed that the amount of class II Ag display, as detected in the DEAD assay, was highest on day 1 postinfection and then declined over time, suggesting that class II Ag processing and presentation occur rapidly after infection. Kaech et al. (60) have shown that whereas the level of LM infection controls the number of CD8+ T cell precursors recruited to respond and subsequently the magnitude of the response, all T cells that are recruited divide extensively. Thus, in our studies the requirement for a longer interval of unmodified infection to generate a maximal CD4+ T cell response may reflect different rates of recruitment between CD4+ and CD8+ T cells in vivo or could reflect a lower in vivo Ag-specific CD4+ T cell precursor frequency.
Secondary expansion of CD4+ T cells after rechallenge of ampicillin-treated immune and control immune groups was similar, as was secondary expansion of CD8+ T cells in the same mice, consistent with previous findings for memory CD8+ T cell responses in BALB/c mice (16). Ampicillin-treated immune and control immune mice also exhibited similar resistance to rLM-OVA rechallenge. A previous study suggested that ampicillin treatment after LM infection resulted in loss of protective immunity against a subsequent challenge (46). This discrepancy may result from several differences in the experimental systems, such as the mouse and LM strains that were used. Additionally, we were able to measure memory and secondary Ag-specific CD4+ and CD8+ T cell responses in ampicillin-treated immune and control immune mice, whereas these analyses were not possible when the earlier study was performed. Other experimental differences were the duration of antibiotic treatment used to truncate LM infection. In contrast to the previous study, we show that in our experimental conditions, prolonged infection is not required for a primary T cell response or protective immunity upon rechallenge. This result has implications for vaccine development, because it indicates that vaccination with live bacterial vectors and treatment with ampicillin allow the generation of protective immunity, with the added potential benefit of improved safety.
Studies comparing the kinetics of primary and secondary T cell responses in the same animal have shown that the contraction phase of the secondary CD8+ T cell response is prolonged compared with that of the primary response (17, 48, 61, 62, 63, 64). Studies by Grayson et al. have demonstrated that memory T cells are more resistant than naive T cells to apoptosis (47) and have elevated levels of the antiapoptotic factor Bcl-2 (65). Consistent with these data, we have demonstrated that the contraction phase of the secondary CD4+ T cell response is also prolonged compared with the contraction phase of the primary response. However, we observed greater contraction of the secondary CD4+ T cell response compared with the secondary CD8+ T cell response by day 13 postinfection. One possible explanation for these results could reflect differences in the APCs for naive and memory T cell populations. Primary responses of naive T cells are generated by interaction with mature DC. Secondary CD8+ T cell responses can presumably be generated through recognition of any infected cell, whereas secondary CD4+ T cell responses are generated only from MHC class II-expressing APCs similar to those that initiated the primary response. Therefore, it is possible that the nature of the APC dictates the kinetics of T cell contraction.
Our data show that the major features of the CD4+ T cell response to LM infection, including magnitude of expansion and onset and rate of contraction, are minimally affected by truncating the duration of infection or Ag display. These results narrow our experimental focus for generating functional T cell memory to early events after infection and, as such, may have important implications for vaccine design.
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Acknowledgments |
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antisera. The expert technical assistance of Katherine Rensberger, Elena Gutierrez, and Rebecca Podyminogen is greatly appreciated. We thank Drs. Vladimir Badovinac and Stanley Perlman for critical review of the manuscript. |
Footnotes |
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1 This work was supported by National Institutes of Health Grants AI42767, AI46653, and AI50073. 
2 Address correspondence and reprint requests to Dr. John T. Harty, Department of Microbiology, University of Iowa, 3-512 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242. E-mail address: john-harty{at}uiowa.edu
3 Abbreviations used in this paper: LM, Listeria monocytogenes; DC, dendritic cell; DEAD assay, direct ex vivo Ag detection assay; ICS, intracellular cytokine staining; LLO, listeriolysin O; rLM-OVA, LM-expressing OVA; PBS-T, PBS with 0.05% Tween 20.
Received for publication January 6, 2004. Accepted for publication August 25, 2004.
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References |
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 N.-L. L. Pham, V. P. Badovinac, and J. T. Harty A Default Pathway of Memory CD8 T Cell Differentiation after Dendritic Cell Immunization Is Deflected by Encounter with Inflammatory Cytokines during Antigen-Driven Proliferation J. Immunol., August 15, 2009; 183(4): 2337 - 2348. [Abstract] [Full Text] [PDF]
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S. L. Colpitts, N. M. Dalton, and P. Scott IL-7 Receptor Expression Provides the Potential for Long-Term Survival of Both CD62Lhigh Central Memory T Cells and Th1 Effector Cells during Leishmania major Infection J. Immunol., May 1, 2009; 182(9): 5702 - 5711. [Abstract] [Full Text] [PDF]
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K.-E. Tseng, C.-Y. Chung, W. S. H'ng, and S.-L. Wang Early Infection Termination Affects Number of CD8+ Memory T Cells and Protective Capacities in Listeria monocytogenes-Infected Mice upon Rechallenge J. Immunol., April 15, 2009; 182(8): 4590 - 4600. [Abstract] [Full Text] [PDF]
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 P. C. Maciag, M. M. Seavey, Z.-K. Pan, S. Ferrone, and Y. Paterson Cancer Immunotherapy Targeting the High Molecular Weight Melanoma-Associated Antigen Protein Results in a Broad Antitumor Response and Reduction of Pericytes in the Tumor Vasculature Cancer Res., October 1, 2008; 68(19): 8066 - 8075. [Abstract] [Full Text] [PDF]
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J. K. Whitmire, N. Benning, B. Eam, and J. L. Whitton Increasing the CD4+ T Cell Precursor Frequency Leads to Competition for IFN-{gamma} Thereby Degrading Memory Cell Quantity and Quality J. Immunol., May 15, 2008; 180(10): 6777 - 6785. [Abstract] [Full Text] [PDF]
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A.-H. Hovav, M. W. Panas, S. Rahman, P. Sircar, G. Gillard, M. J. Cayabyab, and N. L. Letvin Duration of Antigen Expression In Vivo following DNA Immunization Modifies the Magnitude, Contraction, and Secondary Responses of CD8+ T Lymphocytes J. Immunol., November 15, 2007; 179(10): 6725 - 6733. [Abstract] [Full Text] [PDF]
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V. V. Ganusov Discriminating between Different Pathways of Memory CD8+ T Cell Differentiation J. Immunol., October 15, 2007; 179(8): 5006 - 5013. [Abstract] [Full Text] [PDF]
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 D. A. Blair and L. Lefrancois Increased competition for antigen during priming negatively impacts the generation of memory CD4 T cells PNAS, September 18, 2007; 104(38): 15045 - 15050. [Abstract] [Full Text] [PDF]
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H. Hara, I. Kawamura, T. Nomura, T. Tominaga, K. Tsuchiya, and M. Mitsuyama Cytolysin-Dependent Escape of the Bacterium from the Phagosome Is Required but Not Sufficient for Induction of the Th1 Immune Response against Listeria monocytogenes Infection: Distinct Role of Listeriolysin O Determined by Cytolysin Gene Replacement Infect. Immun., August 1, 2007; 75(8): 3791 - 3801. [Abstract] [Full Text] [PDF]
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X. Li, K. K. McKinstry, S. L. Swain, and D. K. Dalton IFN-{gamma} Acts Directly on Activated CD4+ T Cells during Mycobacterial Infection to Promote Apoptosis by Inducing Components of the Intracellular Apoptosis Machinery and by Inducing Extracellular Proapoptotic Signals J. Immunol., July 15, 2007; 179(2): 939 - 949. [Abstract] [Full Text] [PDF]
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F. R. Santori, Z. Popmihajlov, V. P. Badovinac, C. Smith, S. Radoja, J. T. Harty, and S. Vukmanovic TCRbeta Chain That Forms Peptide-Independent Alloreactive TCR Transfers Reduced Reactivity with Irrelevant Peptide/MHC Complex J. Immunol., May 15, 2007; 178(10): 6109 - 6114. [Abstract] [Full Text] [PDF]
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M. O. Kilinc, K. S. Aulakh, R. E. Nair, S. A. Jones, P. Alard, M. M. Kosiewicz, and N. K. Egilmez Reversing Tumor Immune Suppression with Intratumoral IL-12: Activation of Tumor-Associated T Effector/Memory Cells, Induction of T Suppressor Apoptosis, and Infiltration of CD8+ T Effectors J. Immunol., November 15, 2006; 177(10): 6962 - 6973. [Abstract] [Full Text] [PDF]
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J. S. Haring and J. T. Harty Aberrant Contraction of Antigen-Specific CD4 T Cells after Infection in the Absence of Gamma Interferon or Its Receptor Infect. Immun., November 1, 2006; 74(11): 6252 - 6263. [Abstract] [Full Text] [PDF]
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D. M. Lindell, M. N. Ballinger, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Immunologic Homeostasis during Infection: Coexistence of Strong Pulmonary Cell-Mediated Immunity to Secondary Cryptococcus neoformans Infection While the Primary Infection Still Persists at Low Levels in the Lungs J. Immunol., October 1, 2006; 177(7): 4652 - 4661. [Abstract] [Full Text] [PDF]
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 M. Prlic, G. Hernandez-Hoyos, and M. J. Bevan Duration of the initial TCR stimulus controls the magnitude but not functionality of the CD8+ T cell response J. Exp. Med., September 4, 2006; 203(9): 2135 - 2143. [Abstract] [Full Text] [PDF]
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D. I. M. Loeffler, C. U. Schoen, W. Goebel, and S. Pilgrim Comparison of Different Live Vaccine Strategies In Vivo for Delivery of Protein Antigen or Antigen-Encoding DNA and mRNA by Virulence-Attenuated Listeria monocytogenes Infect. Immun., July 1, 2006; 74(7): 3946 - 3957. [Abstract] [Full Text] [PDF]
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 A. Jabbari and J. T. Harty The generation and modulation of antigen-specific memory CD8 T cell responses J. Leukoc. Biol., July 1, 2006; 80(1): 16 - 23. [Abstract] [Full Text] [PDF]
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A. Jabbari and J. T. Harty Secondary memory CD8+ T cells are more protective but slower to acquire a central-memory phenotype J. Exp. Med., April 17, 2006; 203(4): 919 - 932. [Abstract] [Full Text] [PDF]
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B. B. Porter and J. T. Harty The Onset of CD8+-T-Cell Contraction Is Influenced by the Peak of Listeria monocytogenes Infection and Antigen Display Infect. Immun., March 1, 2006; 74(3): 1528 - 1536. [Abstract] [Full Text] [PDF]
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J. K. Whitmire, N. Benning, and J. L. Whitton Precursor Frequency, Nonlinear Proliferation, and Functional Maturation of Virus-Specific CD4+ T Cells. J. Immunol., March 1, 2006; 176(5): 3028 - 3036. [Abstract] [Full Text] [PDF]
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J. S. Haring, V. P. Badovinac, M. R. Olson, S. M. Varga, and J. T. Harty In Vivo Generation of Pathogen-Specific Th1 Cells in the Absence of the IFN-{gamma} Receptor J. Immunol., September 1, 2005; 175(5): 3117 - 3122. [Abstract] [Full Text] [PDF]
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R. Obst, H.-M. van Santen, D. Mathis, and C. Benoist Antigen persistence is required throughout the expansion phase of a CD4+ T cell response J. Exp. Med., May 16, 2005; 201(10): 1555 - 1565. [Abstract] [Full Text] [PDF]
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