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. Author manuscript; available in PMC: 2014 May 9.
Published in final edited form as: J Immunol. 2008 Aug 1;181(3):1700–1709. doi: 10.4049/jimmunol.181.3.1700

IFN-γ Induces the Erosion of Preexisting CD8 T Cell Memory during Infection with a Heterologous Intracellular Bacterium1

Renu Dudani *, Kaja Murali-Krishna †, Lakshmi Krishnan *,‡, Subash Sad *,‡,2
PMCID: PMC4015950  CAMSID: CAMS2224  PMID: 18641306

Abstract

Memory T cells are critical for the control of intracellular pathogens and require few signals for maintenance; however, erosion of established preexisting memory CD8+ T cells has been shown to occur during infection with heterologous viral infections. We evaluated whether this also occurs during infection with various intracellular bacteria and what mechanisms may be involved. We demonstrate that erosion of established memory is also induced during infection of mice with various intracellular bacteria, such as Listeria monocytogenes, Salmonella typhimurium, and Mycobacterium bovis (bacillus Calmette-Guérin). The extent of erosion of established CD8+ T cell memory was dependent on the virulence of the heterologous pathogen, not persistence. Furthermore, when antibiotics were used to comprehensively eliminate the heterologous pathogen, the numbers of memory CD8+ T cells were not restored, indicating that erosion of preexisting memory CD8+ T cells was irreversible. Irrespective of the initial numbers of memory CD8+ T cells, challenge with the heterologous pathogen resulted in a similar extent of erosion of memory CD8+ T cells, suggesting that cellular competition was not responsible for erosion. After challenge with the heterologous pathogen, effector memory CD8+ T cells were rapidly eliminated. More importantly, erosion of preexisting memory CD8+ T cells was abrogated in the absence of IFN-γ. These studies help reveal the paradoxical role of IFN-γ. Although IFN-γ promotes the control of intracellular bacterial replication during primary infection, this comes at the expense of erosion of preexisting memory CD8+ T cells in the wake of infection with heterologous pathogens.

Memory T cells can be maintained in the absence of Ag and MHC (1–3); however, in the face of immune challenge with distinct pathogens, attrition of an otherwise stable memory has been documented (4, 5). However, the factors responsible for establishing the equilibrium between these competing requirements remain unclear. It has been shown that the generation of CD8+ T cell memory is governed by the initial burst size of the effector response (6, 7), which in turn is defined by the Ag burst during the initial phase of infection (8, 9). Following challenge with lymphocytic choriomeningitis virus, 40–60% of CD8+ T cells appear to be virus specific, yet <5% of these cells survive following pathogen clearance (6). With the resolution of the virus infection, a new homeostasis of the memory pool is established but virus-specific CD8+ T cells can remain as high as 10% of the memory pool (5, 6). However, this otherwise stable memory T cell pool is subject to Ag-dependant attrition and a single secondary viral infection can reduce Ag-specific CD8+ T cell numbers by 2- to 5-fold, depending on the viruses involved (5).

Although CD8+ T cells have been considered to play an essential role mainly during viral infection models, they also play an important role in mediating protection against intracellular bacteria such as Listeria monocytogenes (LM)3 (10 –12), Mycobacterium tuberculosis (13), and Salmonella typhimurium (ST) (14, 15). In this study, we use three intracellular bacteria, LM, ST, and Mycobacterium bovis bacillus Calmette-Guérin (BCG), and we contrast the attrition of memory T cells previously observed in viral models (4, 5) with that in our model of heterologous bacterial infections. LM, BCG, and ST are all intracellular bacteria that thrive within macrophages and induce infections that are different in the intensity and duration (16 –18). Following i.v. infection, LM grow rapidly reaching maximum burdens within 72 h, after which the bacteria are rapidly cleared from an immune-competent host and are generally undetectable in the spleen by day 7 (19 –21). BCG, in contrast, induces a chronic infection, the bacterial burden peaks at about 2–3 wk after infection, then declines to a plateau where it remains chronically (22, 23). ST is a highly virulent pathogen that induces gastroenteritis in humans. In the C57BL/6J strain of mice, ST (strain SL1344) induces a lethal infection between days 7 and 10, even when used at doses as low as 102 i.v. In contrast, ST induces a chronic, but nonlethal, infection in 129SvJ or B6.129F1 mice where the infection is usually cleared by days 60 –90.

Our results indicate that the intensity of erosion of preexisting CD8+ T cell memory depends on the virulence of the heterologous pathogen. We also show that effector memory CD8+ T cells are rapidly eliminated during challenge with heterologous pathogens and that IFN-γ plays a principal role in mediating erosion of pre-existing memory CD8+ T cells during challenge with heterologous intracellular bacteria.

Materials and Methods

Intracellular bacteria

BCG (Pasteur) was grown at 37°C under constant shaking in 7H9 medium containing glycerol (0.2%), Tween 80 (0.05%), and albumin-dextrose supplement (10%; Difco Laboratories). At mid-log phase (OD600 = 1.0), bacteria were harvested and frozen at −70°C (in 20% glycerol). CFUs were determined by plating serial dilutions in PBS-T (0.025% Tween 80) on Middlebrook 7H10 solid medium containing glycerol (0.5%) and oleic acid-albumin-dextrose supplement (10%; Difco Laboratories). A listerio-lysin-positive, streptomycin-resistant strain of LM (10403S) was grown in brain-heart infusion (BHI) medium (Difco Laboratories) supplemented with 50 μg/ml streptomycin (Sigma-Aldrich). CFUs were determined by performing serial dilutions in 0.9% NaCl, which were spread on BHI-streptomycin agar plates. Streptomycin-resistant wild-type (WT; SL1344) and aroA mutant of ST were grown as described for LM. At mid-log phase, bacteria were harvested and frozen in 20% glycerol and stored at −70°C. OVA-expressing LM (LM-OVA), as described previously (24), was grown to OD600 nm = 0.4. The bacteria were grown in BHI medium and stored as described above.

Mice and immunizations

C57BL/6 and 129x1SvJ mice were obtained from The Jackson Laboratory. B6129F1 mice were generated in house by mating 129x1SvJ female mice with C57BL/6 male mice. OT-1 TCR-transgenic mice were obtained from The Jackson Laboratory. Mice were maintained at the Institute for Biological Sciences (National Research Council of Canada, Ottawa, Canada) in accordance with the guidelines of the Canadian Council on Animal Care. For immunization with LM-OVA, frozen stocks were thawed and diluted in 0.9% NaCl. Mice were inoculated with 1 × 104 organisms suspended in 200 μl of 0.9% NaCl via the lateral tail vein (i.v.). In some experiments, mice were injected first with 104 OT-1 CD8+ T cells (i.v.) and challenged a few days later with LM-OVA (103, i.v.). For immunization with a particulate Ag, mice were injected s.c. with OVA (15 μg in 100 μl of PBS) entrapped in liposomal vesicles composed of archaebacterial lipids (OVA archaeosomes) (25). Thirty days after the last injection, mice were challenged i.v. with BCG, LM, or ST. Age-matched control mice were injected with PBS.

Assessment of bacterial burden in spleen

Single-cell suspensions from infected mice were tweezed in RPMI 1640. CFUs were determined by plating 100-μl aliquots of serial 10-fold dilutions in 0.9% saline on appropriate plates as above. For spleen cells from BCG-challenged mice, the dilutions were prepared in PBS-T (0.025% Tween 80). One hundred-microliter samples of these dilutions were spread on Middlebrook 7H10 solid medium as before. Plates were incubated for 24 h at 37°C in the case of spleens from LM- and ST-challenged mice and for 21–30 days in the case of spleens from BCG-challenged mice.

Isolation of cells

Lymphoid (spleen) and nonlymphoid organs (lungs, liver, and brain) were removed. Nonlymphoid organs were removed after perfusion of organs with 40 ml of PBS injected through the heart artery. Single-cell suspensions were prepared by tweezing the pooled organs (n = 2–3) between the frosted ends of two sterile glass slides in RPMI 1640. Cells were subsequently passed through Falcon 2360 cell strainers (BD Labware), centrifuged, and resuspended in RPMI 1640 and 8% FBS supplemented with 50 μg/ml gentamicin (Life Technologies). Cells from nonlymphoid organs were obtained after separation on Percoll gradients (26).

ELISPOT assay

Enumeration of IFN-γ-secreting cells was done by ELISPOT assay (27). Briefly, spleen cells were incubated in anti-IFN-γ Ab-coated ELISPOT plates, varying the number of spleen cells from immunized mice to achieve a final cell density of 5 × 105/well using feeder cells from unimmunized mice. These cultures were established in RPMI 1640 plus 8% FBS. Cells were stimulated with OVA257–264 (10 μg/ml) supplemented with IL-2 (1 ng/ml; unless otherwise indicated) and incubated for 48 h at 37°C/8% CO2. The cells were then lysed with H2O, the plates were washed (PBS plus Tween 20), incubated with the biotinylated secondary Ab (4°C, overnight), followed by avidin-peroxidase conjugate (room temperature, 2 h). Spots were revealed using diaminobenzidine.

Flow cytometry

At various time intervals after infection, aliquots of spleen cells (10 × 106) were incubated in 200 μl of PBS plus 1% BSA (PBS-BSA) with anti-CD16/32 at 4°C. After 10 min., cells were stained with PE-H-2KbOVA257–264 tetramer and various Abs (anti-CD8, anti-CD62L, and anti-CD44) for 30 min at room temperature. All Abs were obtained from BD Biosciences. PE-H-2KbOVA257–264 tetramer was obtained from Beckman Coulter. Cells were washed with PBS and fixed in 0.5% formaldehyde and acquired on a BD Biosciences FACSCanto analyzer. To measure apoptotic commitment of OVA-specific CD8+ T cells, annexin V staining was performed using a Apoptosis Detection Kit from BD Biosciences. Briefly, spleen cells (5 × 106) were stained with anti-CD8 Ab and OVA tetramer as described above. This was followed by washing with PBS and staining in binding buffer with annexin V-FITC for 15 min at room temperature One million events were acquired on a BD FACSCanto flow cytometer.

Cytotoxicity assays

Single-cell suspensions from pooled spleens (n = 3) of immunized mice were prepared as described above. After washing, spleen cells (30 × 106) from various experimental groups were incubated with 5 × 105 irradiated (10,000 rad) Ag-bearing target cells (EG7 cells) in 10 ml of RPMI 1640 plus 8% FBS. Cultures contained 0.1 ng/ml IL-2 and were placed in 25-cm2 tissue culture flasks (Falcon; BD Biosciences) and kept upright. After 5 days (37°C, 8% CO2), cells were harvested from the flasks, washed, counted, and used as effectors in a standard 51Cr release CTL assay. EL4 target cells (1 × 106/ml) were either incubated with medium or with OVA257–264 (10 μg/ml) for 2 h before labeling with radioactive chromium. For labeling, 5 × 106 target cells (EL4 and EL4 plus OVA257–264) were incubated with 50 μl of 51Cr (100 μCi) and 25 μl of RPMI 1640 plus 8% FBS medium. After 45 min, targets were washed twice, various ratios of effectors and targets were cocultured for 4 h in 96-well round-bottom tissue culture plates, and the supernatants were collected and radioactivity was detected by gamma counting. The percent cytotoxicity was calculated using the formula: 100×[(cpmexperimental-cpmspontaneous)/(cpmtotal-cpmspontaneous)].

Assessment of cytokine expression by quantitative RT-PCR

Harvested spleens were snap frozen in a dry ice/100% ethanol bath. Total RNA was extracted using a Qiagen RNeasy Mini Kit according to the manufacturer’s instructions along with rapid mechanical lysis. Total RNA from homogenates was extracted according to the manufacturer’s instructions (Roche Applied Science). RNA was made linear at 65°C for 5 min and cDNA was synthesized in a 40-μl reaction volume containing, 1.5 μl of AncT Primers (100 pmol/μl), 8 μl of 5× First Strand Buffer, 4 μl of DTT (100 mM), 5 μl of dNTPs (5 mM), 1 μl of RNase OUT (40 U/μl), 2 μl of Superscript II (200 U/μl; Invitrogen), and 15 μl of RNA template. Reverse transcription was performed in a Thermo-Cycler 9700 (Applied Biosystems) at 42°C for 15 min and 45°C for 2 h. RNA template was hydrolyzed with 1 M NaOH at 65°C for 5 min and neutralized with 1 M HCl cDNA was purified using Microcon YM-30 centrifugal filter units (Millipore). The number of amplicons was measured by quantitative real-time PCR using gene-specific primers and qPCR SYBR Green Supermix (ABgene). Primers were designed using Primer Express 2.0. β-Actin was used as an internal reference control. Ten-fold dilutions of cDNA were used as template to generate the standard curve for each primer-template set (1×, 1/10×, 1/100×, 1/1000×). This standard curve was run along with triplicate reactions of the uncharacterized samples. PCR was performed in sealed tubes in a 96-well microtiter plate in an Applied Biosystems Prism 7000 thermocycler. The 25-μl reaction consisted of 12.5 μl of qPCR SYBR Green Supermix, 2.5 μl of primer mix (1.5 pmol/μl each), and 10 μl of template. Thermal conditions were as follows: activation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Fluorescence was measured during the annealing step and plotted against the amplification cycle. Relative quantitative analysis of the data was extrapolated from the standard curve. Primer efficiencies were between 100 and 98%.

Results

Erosion of established CD8+ T cell memory in multiple organs

OVA-specific memory CD8+ T cells were generated by infecting C57BL/6J mice with LM-OVA. Thirty days later, mice were injected with PBS or with BCG (105, i.v.). At day 60, spleens, lungs, livers, and brains of mice were removed and the relative numbers of OVA-specific CD8+ T cells were enumerated. In all of the organs tested, erosion of preexisting OVA-specific memory CD8+ T cells was noted in mice that were challenged with BCG (Fig. 1). These results indicate that infection of mice with BCG results in a comprehensive erosion of preexisting CD8+ T cell memory.

FIGURE 1.

FIGURE 1

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Challenge with a heterologous intracellular bacterium causes comprehensive erosion of preexisting CD8+ T cell memory. OVA257–264-specific memory CD8+ T cells were generated in C57BL/6J mice after injection with 104 CD8+ T cells from naive OT-1 TCR- transgenic mice followed by subsequent infection with 104 LM-OVA. On day 30, mice were injected with PBS or BCG (105, i.v.). On day 60, various organs (indicated in this figure) were removed from groups of mice. Cell suspensions from lungs, livers, and brains were obtained after separation on Percoll gradients. Cells were stained with H-2KbOVA257–264 tetramer and Abs against CD8 and CD44, and the relative numbers of OVA-specific CD8+ T cells were enumerated. Representative data of three experiments are shown and each experimental group consisted of three mice.

Dose of the pathogen influences the extent of erosion of CD8+ T cell memory

To determine whether the extent of erosion of preexisting CD8+ T cell memory can be enhanced by increasing the dose of BCG, we challenged groups of LM-OVA-immunized mice with 105 or 106 doses of BCG. Thirty days after BCG challenge, the relative numbers of OVA-specific CD8+ T cells were enumerated in the spleens. As is indicated in Fig. 2, challenge with a 106 dose of BCG resulted in an even greater (>10-fold) erosion of OVA-specific CD8+ T cell memory.

FIGURE 2.

FIGURE 2

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Dose of the heterologous intracellular bacterium influences the extent of memory erosion. C57BL/6J mice were infected with LM-OVA (104, i.v.). On day 30, mice were challenged with various doses of BCG or PBS. On day 60, spleens were removed from the various groups of mice and the numbers of OVA-specific IFN-γ-secreting CD8+ T cells were evaluated by ELISPOT assay. Number of spots per 1 × 106 spleen cells is indicated. Representative data of two experiments are shown and each experimental group consisted of three mice.

Multiple intracellular bacteria induce erosion of CD8+ T cell memory

We determined whether preexisting memory CD8+ T cells would be susceptible to erosion after challenge with intracellular pathogens that differ in pathogenesis, virulence, and inflammation induced. Since ST causes a rapid, lethal infection in C57BL/6J mice, we performed these experiments in B6.129F1 mice that are resistant to ST infection. OVA-specific memory CD8+ T cells were generated after injecting mice with the potent adjuvant system archaeosomes containing OVA. Mice were subsequently challenged with a low dose (103) of LM, ST, or BCG. Infection of mice with doses higher than 103 of ST causes fatality even in resistant mice. Control mice received PBS. At various time intervals after the pathogen challenge, spleens were removed and the relative numbers of OVA-specific CD8+ T cells were enumerated. Within 7 days of pathogen challenge, erosion of OVA-specific CD8+ T cell memory was noted in the case of LM- and ST-infected groups (Fig. 3, A and B). BCG, at this low dose, induced only a marginal decline in the numbers of OVA-specific memory CD8+ T cells. Although ST and BCG induce a chronic infection, LM induces an acute infection that is cleared within the first 5 days (Fig. 3C). BCG induces a more chronic infection relative to ST. These results indicate that the erosion of established CD8+ T cell memory occurs during infection with different pathogens involving highly diverse host-pathogen interactions.

FIGURE 3.

FIGURE 3

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Erosion of preexisting CD8+ T cell memory occurs during infection with various intracellular bacteria. B6.129F1 mice were injected with 104 CD8+ T cells from naive OT-1 TCR-transgenic mice. Immediately thereafter, mice were injected s.c. with OVA entrapped into archaeosomes (10 μg of OVA/mouse). On day 30, mice were challenged with 103 LM, ST, or BCG (i.v.). Controls were injected with PBS. At various time intervals thereafter, spleens were removed from the various groups of mice and stained with OVA tetramers and Abs against CD8 and CD44. Representative profile is shown at day 60 after challenge (A). Numbers in A represent the percentage of OVA-tetramer+ cells among gated CD8+T cells. Change in the numbers of OVA-specific CD8+ T cells after challenge was evaluated kinetically (B). Bacterial burden was evaluated in the spleens at various time intervals (C). Representative data of two experiments are shown and each experimental group consisted of more than three mice.

Infection with intracellular bacteria does not cause generalized depletion of T cells

We evaluated the relative numbers of naive and Ag-experienced CD8+ T cells in mice infected with various intracellular bacteria. PBS-injected mice served as controls. There was a progressive increase in the relative numbers of CD44highCD8+ T cells in infected mice, which is expected since CD8+ T cells would be primed against pathogens (Fig. 4). However, the overall numbers of naive and memory CD8+ T cells (irrespective of antigenic specificity) did not undergo dramatic changes.

FIGURE 4.

FIGURE 4

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Challenge with heterologous pathogen does not cause global depletion of T cells. C57BL/6J mice were injected with PBS or LM (103, i.v.), ST (103, i.v.), or BCG (105, i.v.). At various time intervals, spleens were removed from the various groups of mice and the relative numbers of naive (CD44low) and memory (CD44high) CD8+ T cells were evaluated. Representative data of five experiments are shown and each experimental group consisted of three mice.

Pathogen virulence influences the extent of erosion of CD8+ T cell memory

To specifically evaluate the influence of pathogen virulence on the erosion of preexisting memory CD8+ T cells, we evaluated erosion induced by the WT strain of ST vs the aroA mutant of ST. The aroA mutant of ST lacks the enzyme aromase that is required for metabolizing aromatic compounds and induces a nonlethal infection in C57BL/6J mice (Fig. 5A). In B6.129F1 mice, both the WT and the aroA mutant induce a chronic infection; however, infection with the WT results in increased bacterial burden (Fig. 5B) and inflammation (Fig. 5C). Challenge of OVA archaeosome-injected mice with the WT ST induced greater erosion of memory in comparison to that induced by a challenge with the aroA mutant of ST (Fig. 5D).

FIGURE 5.

FIGURE 5

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Pathogen virulence influences the extent of erosion of preexisting CD8+ T cell memory. C57BL/6J (A) or B6.129F1 (B) mice were infected i.v. with 103 WT or aroA mutant of ST. At various time intervals, the relative bacterial burdens (A and B) and the splenic cell numbers (C) were evaluated. OVA-specific CD8+ T cell memory was generated in B6.129F1 mice by injecting them with OVA archaeosomes (10 μg of OVA/mouse) on days 1 and 30. On day 60, some mice were challenged with 103 WT or aroA mutant of ST. At various time intervals after ST challenge, spleens were removed and stained with OVA tetramers and Abs against CD8 and CD44. The numbers of OVA-specific CD8+ T cells were enumerated (D). Representative data of three experiments are shown and each experimental group consisted of three mice.

Erosion of memory also occurs during repeat infection in the absence of Ag

Rather than inducing erosion after challenging mice with the heterologous pathogen, we addressed the question whether erosion of preexisting CD8+ T cell memory would occur after mice are challenged with the same pathogen in the absence of Ag. To this end, we infected mice first with LM-OVA and challenged these mice with LM 30 days later. PBS-injected mice served as controls. On day 60, spleens were removed and the numbers and cytolytic activity of OVA-specific CD8+ T cells was enumerated. Challenge of mice with the same pathogen (LM) in the absence of Ag (OVA) resulted in a significant erosion of preexisting OVA-specific CD8+ T cell memory (Fig. 6, A–C).

FIGURE 6.

FIGURE 6

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Erosion of preexisting CD8+ T cell memory occurs even during repeat infection with the same pathogen in the absence of Ag. OVA-specific CD8+ T cell memory was induced in C57BL/6J mice by infecting them with LM-OVA (104, i.v.). On day 30, mice were challenged with LM (105, i.v.) and the cytolytic activity of OVA-specific CD8+ T cells was evaluated on day 60 (A). To evaluate the influence on the relative numbers of OVA-tetramer+ cells, OVA-specific memory CD8+ T cells were generated in mice that were initially parked with 104 OT-1 TCR-transgenic CD8+ T cells and challenged with LM-OVA. Thirty days later, a group of mice was challenged with LM and the relative numbers of OVA-specific CD8+ T cells were enumerated at day 60 (B and C). Numbers in B represent the percentage of OVA-tetramer+ cells among gated CD8+T cells. Representative data of two experiments are shown and each experimental group consisted of three mice.

Erosion of established CD8+ T cell memory is permanent

It was possible that erosion of established CD8+ T cell memory was only a temporary phenomena that was due to persistence of the pathogen and consequent inflammation. To address this question, we generated OVA-specific CD8+ T cell memory in mice by injecting them with OVA archaeosomes. At day 30, mice were injected with PBS or challenged with ST or BCG. From day 60 onward, a group of ST-challenged mice received ampicillin (10 mg/ml) and a group of BCG-challenged mice received isoniazid and rifampicin (300 μg/ml each) in the drinking water continuously until day 90. Treatment of ST- or BCG-infected mice with antibiotics did not restore the numbers of OVA-specific memory CD8+ T cells (Fig. 7, A and D). Antibiotic treatment curtailed the inflammation back to control levels (Fig. 7, B and E) and reduced the bacterial burden to undetectable levels (Fig. 7, C and F).

FIGURE 7.

FIGURE 7

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Erosion of preexisting CD8+ T cell memory is permanent. OVA-specific CD8+ T cell memory was generated in B6.129F1 mice by injecting them with OVA archaeosomes (10 μg of OVA/mouse) on day 1. On day 30, some mice were challenged with 103 WT ST or 105 BCG (i.v.). From day 60 onward, a group of ST-challenged mice received ampicillin (10 mg/ml) and a group of BCG-challenged mice received isoniazid and rifampicin (300 μg/ml each) in the drinking water continuously until day 90. On day 90, spleens were removed from the various groups of mice and the numbers of OVA-specific IFN-γ-secreting CD8+ T cells were evaluated by ELISPOT assay (A and D). The relative numbers of spleen cells (B and E) and bacterial burden (C and F) in the spleens of various groups of mice was evaluated at day 90. Representative data of two experiments are shown and each experimental group consisted of three mice.

Fraction of memory CD8+ T cells that are eroded remains constant

We determined whether the fraction of established memory CD8+ T cells that are eroded during a heterologous pathogen challenge depended on the relative numbers of preexisting memory CD8+ T cells. To this end, various numbers of OT-1 TCR-transgenic CD8+ T cells were injected into mice that were challenged subsequently with LM-OVA. This results in the generation of varying numbers of OVA-specific memory CD8+ T cells in mice. At day 30, mice were challenged with BCG and the erosion of OVA-specific CD8+ T cells was enumerated at day 60. Irrespective of the relative numbers of preexisting OVA-specific memory CD8+ T cells, the fraction of memory CD8+ T cells that were eroded after BCG challenge remained constant (Fig. 8). Approximately 3-fold reduction in the numbers of preexisting OVA-specific memory CD8+ T cells was noted after BCG challenge. Thus, when OVA-specific memory CD8+ T cells are present in high numbers (~1 in every 5 CD8+ T cells) or relatively lower numbers (~1 in every 50 CD8+ T cells), they display similar susceptibility to erosion during a heterologous challenge.

FIGURE 8.

FIGURE 8

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The extent of erosion of memory CD8+ T cells does not depend on their initial numbers. C57BL/6J mice were injected with different numbers of CD8+ T cells from OT-1 TCR-transgenic mice and infected with LM-OVA. On day 30, some mice were challenged with BCG (105). On day 60, spleens were removed from the various groups of mice. Spleen cells were stained with H-2KbOVA257–264 tetramer and Abs against CD8 and CD44 and the relative numbers of OVA-specific CD8+ T cells were enumerated. Representative data of three experiments are shown and each experimental group consisted of three mice.

Multiple boosting of memory CD8+ T cells does not prevent erosion

It has been previously reported that boosting of memory CD8+ T cells in vivo results in their increased expression of the antiapoptotic mediators such as Bcl2 which results in their decreased contraction subsequently (28). We therefore determined whether memory CD8+ T cells that were boosted multiple times were uniquely less susceptible to erosion. Mice were injected with LM-OVA at monthly intervals. At the time of the last (fifth) injection, LM-OVA was also injected into a new group of naive mice. Thirty days after the last injection, mice were injected with PBS or BCG and the influence on preexisting OVA-specific memory CD8+ T cells enumerated. Whether mice were injected only once or five times with LM-OVA, OVA-specific memory CD8+ T cells displayed similar susceptibility to erosion after BCG challenge (Fig. 9).

FIGURE 9.

FIGURE 9

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Repeated boosting of memory CD8+T cells does not prevent their subsequent erosion. OVA257–264-specific memory CD8+ T cells were generated in C57BL/6J mice after injection with 104 CD8+ T cells from naive OT-1 TCR-transgenic mice followed by subsequent infection with LM-OVA (104, i.v.). Mice were boosted with LM-OVA (105, i.v.) every 30 days. At the time of the fifth injection, LM-OVA was also injected into a new group of OT-1 parked naive C57BL/6J mice. Thirty days after the last injection, mice were injected with PBS or BCG. After an additional 30 days, spleens were removed from mice and spleen cells were stained with OVA tetramers and Abs against CD8 and CD44. The relative numbers of OVA-specific CD8+ T cells were enumerated (A and B). Numbers in A represent the percentage of OVA-tetramer+ cells among gated CD8+T cells. Representative data of two experiments are shown and each experimental group consisted of four mice.

Effector memory CD8+ T cells are rapidly eliminated

We reasoned that if apoptosis is responsible for erosion of preexisting memory CD8+ T cells during heterologous pathogen challenge, then the cells should bind to annexin V. Seven days after challenge of mice with the heterologous pathogen ST, erosion of preexisting OVA-specific memory CD8+ T cells was noted (Fig. 10A). Spleen cells were stained with annexin V, OVA tetramer and anti-CD8 Ab at day 5 after ST infection. Increased numbers of OVA-specific CD8+ T cells bound to annexin V, indicating apoptotic commitment (Fig. 10B). We also stained the cells with Abs against CD62L and CD44 and noted that the majority of CD62Llow OVA-specific memory CD8+ T cells were eliminated in mice challenged with ST, implying that effector memory cells were rapidly eliminated during infection with heterologous pathogen challenge (Fig. 9, C and D).

FIGURE 10.

FIGURE 10

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Effector memory CD8+T cells are highly susceptible to erosion. OVA257–264-specific memory CD8+ T cells were generated in B6.129F1 mice after injection with 104 CD8+ T cells from naive OT-1 TCR-transgenic mice followed by two injections with OVA archaeosomes (days 1 and 30). On day 60, groups of mice were injected with PBS or challenged with ST. On day 67, spleens were removed from the various groups of mice and the numbers of OVA-specific CD8+ T cells were enumerated after staining spleen cells with OVA tetramers and anti-CD8 and anti-CD44 Abs (A). Numbers in A represent the percentage of OVA- tetramer+ cells among gated CD8+T cells. The numbers of apoptotic OVA-specific CD8+ T cells were enumerated at day 65 after staining with OVA tetramer, anti-CD8 Ab, and annexin V (B). Cells were also stained with anti-CD62L and anti-CD44 Abs to discriminate between central and effector memory cells (C and D). Numbers in B and C indicate the percentages of cells among gated OVA-tetramer+CD8+ T cells. Representative data of three experiments are shown and each experimental group consisted of three mice.

IFN-γ induces the erosion of CD8+ T cell memory

We sought to determine the mechanism responsible for causing erosion of preexisting CD8+ T cell memory during challenge with heterologous intracellular bacterium. Control WT mice or mice with deficiency in key inflammatory mediators were injected with LM-OVA. Antibiotics were given at 48 h to control LM-OVA burden. Mice were boosted on day 30 with LM-OVA and challenged with BCG or ST (ΔaroA) on day 60. Thirty days later, the influence on OVA-specific memory CD8+ T cells was enumerated. In the absence of IFN-γ, erosion of preexisting OVA-specific CD8+ T cell memory was completely abrogated, indicating that IFN-γ is a key mediator that promotes erosion of preexisting CD8+ T cell memory during infection with heterologous intracellular bacterium (Fig. 11). Erosion of preexisting CD8+ T cell memory occurred normally in mice that were deficient in inducible NO synthase 2 or IFN-α receptor (Fig. 11). Erosion also proceeded normally in mice that lacked perforin or had a mutation in FasL (our unpublished observations).

FIGURE 11.

FIGURE 11

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IFN-γ plays a key role in causing the erosion of preexisting memory CD8+ T cells. WT, IFN-γ-deficient, inducible NO synthase 2-deficient and IFN-Rγ-deficient C57BL/6J mice were infected with 103 LM-OVA. Ampicillin (10 mg/ml) was provided in the drinking water from day 2 until day 10. Mice were boosted with LM-OVA (105) on day 30. On day 60, groups of mice were challenged with BCG and ST (ΔaroA) or injected with PBS. On day 90, spleens were removed from the various groups of mice and the relative numbers of OVA-specific CD8+ T cells were enumerated after staining spleen cells with OVA tetramers and anti-CD8 and anti-CD44 Abs. Representative data of two experiments are shown and each experimental group consisted of four mice.

IFN-γ, but not IFN-α, is highly expressed during infection with intracellular bacteria

We sought to determine the relative levels of IFN-γ and IFN-α during infection with the intracellular bacteria. This was achieved by performing quantitative RT-PCR on spleen samples and values were normalized to β-actin. Splenic expression of IFN-γ correlated to the duration of infection and the virulence of the pathogen with spleens of LM- and ST- infected mice expressing higher levels of IFN-γ in comparison to BCG-infected spleens (Fig. 12). None of the infected spleens expressed any significant levels of IFN-α.

FIGURE 12.

FIGURE 12

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IFN-γ is expressed at higher levels during infection with intracellular bacteria. B6.129F1 mice were injected with PBS or infected with LM (103, i.v.), ST (103, i.v.), or BCG (105, i.v.). At various time intervals, spleens were removed and snap frozen in a dry ice ethanol bath. Total RNA was extracted and 5 μg of RNA was taken to make cDNA. The amount of RNA for IFN-γ and IFN-α and β-actin was measured using the SYBR Green method of quantification as outlined in Materials and Methods. Values represent the levels of cytokine mRNA relative to β-actin mRNA. Representative data of two experiments are shown and each experimental group consisted of three mice.

Erosion of established memory results in compromised protection against pathogen rechallenge

We tested whether erosion of established memory has any serious consequences. To this end, we induced memory in mice by infecting them with LM-OVA, followed by erosion in a group of mice with ST challenge. After memory erosion, ST burden was obliterated using antibiotics. We then tested the ability of mice to resist a challenge with a lethal dose of LM-OVA. As is indicated in Fig. 13, erosion of memory by ST results in massive impairment in resistance to a rechallenge with LM-OVA. These results clearly indicate that erosion of established T cell memory during heterologous pathogens can have serious consequences.

FIGURE 13.

FIGURE 13

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Erosion of established T cell memory compromises protection against pathogen rechallenge. B6.129F1 mice were injected with PBS or infected with LM-OVA (102, i.v.). Thirty days later, one group of LM-OVA-infected mice were challenged with ST (103, i.v.). From day 60 onward, mice received ampicillin (10 mg/ml) in drinking water. Mice were put on normal drinking water from day 90 onward. On day 105, all of the groups of mice were challenged with a lethal dose of LM-OVA (105, i.v.) and the bacterial burden in the spleen was evaluated 3 days later. Representative data of two experiments are shown and each experimental group consisted of three to four mice.

Discussion

Understanding how an ongoing immune response to an infection alters responses generated to previously encountered pathogens is important since a host can potentially be challenged with multiple pathogens. There is a clear selective advantage for both the maintenance of memory against life-threatening pathogens, while also having a capacity to respond to new challenges. Hence, a balance must be struck between the acquisition of new memory and the maintenance of previously generated memory (5). It has been previously shown that viral infections induce erosion of established preexisting CD8+ T cell memory (4, 5). This erosion of established memory is not restricted to viral challenges as we have previously reported that infection of mice with the intracellular bacterium BCG also results in erosion of established CD8+ T cell memory (29). Several conclusions can be drawn from our results. We show that erosion of established CD8+ T cell memory occurs during infection with multiple intracellular bacteria and that the extent of the loss of memory depends on the virulence of the pathogen. We also show that erosion of memory that occurs during infection with intracellular bacteria is mediated by IFN-γ and targets effector memory cells rapidly.

Erosion of CD8+ T cell memory was not restricted to only one site, implying that the erosion of CD8+ T cell memory is comprehensive and not due to differential homing of memory cells to other organs during infection with heterologous pathogens. Furthermore, erosion of CD8+ T cell memory was irreversible as removal of the heterologous chronic pathogens (ST and BCG) with antibiotics did not restore the relative numbers of memory CD8+ T cells. We have previously reported that such erosion of CD8+ T cell memory results in compromised protection against a challenge with OVA-expressing tumor cells highlighting the functional consequences of erosion of OVA-specific CD8+ T cell memory (29). Our results now indicate that erosion of established memory also results in compromised protection against pathogen rechallenge.

In the viral infection models, a 2- to 5-fold drop in CD8+ T cell memory was reported after challenge with heterologous viruses (5). We show that the extent of erosion depends on the virulence of the pathogen. BCG, ST, and LM vary in their intracellular habitat, replication rate, virulence mechanism, type of infection caused, and the nature of inflammation induced (16). Of the three pathogens that we used, ST is the most virulent and consequently resulted in the maximal erosion of preexisting CD8+ T cell memory, whereas BCG is the least virulent pathogen which induced the least erosion of memory. However, BCG is the most chronic pathogen, as detectable burden is noticeable even at 6 mo postinfection, whereas ST burden declines after day 90 (17, 18). LM infection lasts for <7 days (21) and results in erosion that is greater than that induced by BCG. Taken together, these results indicate that virulence, rather than the chronicity, of the pathogen is the key factor in terms of the extent of erosion that can occur during infection with heterologous pathogens. We have shown here that the erosion of established CD8+ T cell memory can occur even during a repeat infection with the same pathogen in the absence of Ag. This suggests that the pathogen that induces erosion of preexisting memory need not be heterologous to the original pathogen. This has implications for chronic pathogens such as HIV where ongoing mutations in HIV Ags will result in erosion of established CD8+ T cells against HIV.

In this work, attrition was determined by functional assays such as: Ag-specific CTL activity, IFN-γ-secretion, and tetramer staining. Our data indicate that OVA-specific CD8+T cells are likely to be deleted after bacterial challenge. If significant numbers of Ag-specific T cells were present in LM plus BCG- infected mice and merely anergized, then the IL-2 and Ag used during the CTL restimulations and the ELISPOT assays would likely have contributed to their release from that state (30 –32). We have previously reported enhanced IFN-γ response from purified T cells in the BCG-challenged mice when stimulated with anti-CD3, but reduced LM-specific IFN-γ response when stimulated with LM-Ags, arguing against anergy and indicating that LM-specific T cells are deleted during BCG challenge (29).

There is considerable controversy with respect to the influence of BCG on immune responses with some suggesting that BCG is a potent adjuvant for the induction of the T cell response to recombinant Ags (33, 34), but on the other hand others have associated BCG with suppressive effects (35–41). Similar suppressive effects have been reported in ST infection models (42). Our previous observations indicate that BCG is neither a suppressive nor potent inducer of CD8+ T cell responses (24, 43). Evaluation of immune responses in vivo vs in vitro can give contrasting results depending on the assays used and relying solely on in vitro results can be misleading (44). During infection with intracellular bacteria, such as BCG, numerous inflammatory cytokines and immunoactive compounds are induced chronically (36, 45, 46). These inflammatory mediators may influence different facets of immune responses.

Memory CD8+ T cells have been shown to undergo less contraction in response to antigenic stimulation in vivo (28). Furthermore, multiple boosting of memory CD8+ T cells has been shown to result in an increased level of antiapoptotic mediator Bcl2 (28). Our results indicate that memory CD8+ T cells that underwent multiple boosting were equally susceptible to erosion after infection with a heterologous pathogen. It is quite conceivable that the contraction of an ongoing CD8+ T cell response and the erosion of established memory CD8+ T cells are phenomena that involve distinct mechanisms.

Our results indicate that erosion of memory CD8+ T cells occurred irrespective of the relative numbers of preexisting memory CD8+ T cells present before encounter with the heterologous pathogen. This suggests that the erosion of memory may not be due to clonal competition, but due to an active process during which a fraction of memory CD8+ T cells are deleted (47). Intuitively, the need for an attrition process to accommodate the generation of memory responses to new immune challenges is clear. Apoptosis has been considered as a principal mechanism responsible for the attrition of effectors (5, 6, 48) allowing survival of a small percentage of cells as memory cells. In the viral attrition model, apoptosis of preexisting memory CD8+ T cells was dependent on the expression of IFN-α/β during subsequent viral challenges (49). Our study indicates that during challenges with intra-cellular bacteria, IFN-γ induces the erosion of preexisting memory CD8+ T cells. This suggests that multiple mechanisms may operate to induce erosion of established CD8+ T cell memory during infection with heterologous pathogens. IFN-α/β is expressed at high levels during viral infections where it plays a key role (50, 51). In bacterial infections, IFN-α/β is expressed minimally and plays little role (50, 51). Our data also indicate little IFN-γ expression during infection with intracellular bacteria. High levels of IFN-γ are detectable in the serum of BCG-infected mice (52), and IFN-γ plays a key role in the pathogenesis of BCG and other bacterial infections (53–55). BCG has been reported to induce apoptosis of T cells (37, 56), and IFN-γ has been implicated in the induction of apoptosis of effector T cells during infections with BCG (57), LM (58), Toxoplasma gondii (59), and Trypanosoma cruzi (60). In these models, apoptosis appears to be induced by IFN-γ as the Ag levels decline. In our model, the BCG-induced IFN-γ response and subsequent apoptosis occurs in the absence of LM-Ags.

Which cells within the memory T cell pool are eliminated in the absence of their cognate Ag may depend on a variety of factors, including the phase of cell cycle and/or the cell’s activation status, in addition to the balance between pro- and antiapoptotic regulatory factors in the T cells, before their encounter with the secondary BCG infection. Our results indicate that effector memory CD8+ T cells are rapidly eliminated during infection with heterologous intracellular bacteria. Indeed, recently effector and central memory subsets of CD4+ T cells have been shown to display different signaling mechanisms leading to their differential survival (61). Given the complexity of the phenotypes of memory T cells (62), it is not clear whether effector memory cells are the only subset of memory CD8+ T cells that are targeted for erosion during a heterologous pathogen challenge. When mice are challenged with a heterologous pathogen at a very late stage (day 90) when a majority of the preexisting memory CD8+ T cells display mainly the central memory phenotype, erosion of memory is still noted (our unpublished data). It is therefore conceivable that although both subsets may be susceptible to erosion in the long term, effector memory cells may be susceptible to deletion more rapidly during infection with the heterologous pathogen.

The complexities of selecting and ordering bacterial pathogens is illustrated by the reverse infection model involving BCG plus LM (63). Infection of mice first with BCG and then challenging them with LM results in poor T cell priming against LM. In this case, the chronic inflammation induced by BCG was sufficient to prevent the growth of LM in vivo, resulting in attenuation of T cell priming against LM. This suggests that pathogens that induce significant inflammation not only cause erosion of preexisting memory CD8+ T cells, but the inflammation induced also restricts T cell priming and hence memory development against pathogens that are encountered later. Thus, any time a pathogen is introduced in the host, it perturbs past and future immune responses.

Footnotes

1

This work was supported by a grant from the Canadian Institutes of Health Research.

3

Abbreviations used in this paper: LM, Listeria monocytogenes; ST, Salmonella typhimurium; BCG, Mycobacterium bovis; BHI, brain-heart infusion; WT, wild type.

Disclosures

The authors have no financial conflict of interest.

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