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The Journal of Immunology, 2007, 179, 211 -220
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Reduced Antigen Load In Vivo, Rather Than Weak Inflammation, Causes a Substantial Delay in CD8+ T Cell Priming against Mycobacterium bovis (Bacillus Calmette-Guérin)1

Marsha S. Russell*,{dagger}, Monica Iskandar*, Oksana L. Mykytczuk*, John H. E. Nash*, Lakshmi Krishnan*,{dagger} and Subash Sad2,*,{dagger}

* National Research Council–Institute for Biological Sciences and {dagger} Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regardless of the dose of Ag, Ag presentation occurs rapidly within the first few days which results in rapid expansion of the CD8+ T cell response that peaks at day 7. However, we have previously shown that this rapid priming of CD8+ T cells is absent during infection of mice with Mycobacterium bovis (bacillus Calmette-Guérin (BCG)). In this study, we have evaluated the mechanisms responsible for the delayed CD8+ T cell priming. Because BCG replicates poorly and survives within phagosomes we considered whether 1) generation of reduced amounts of Ag or 2) weaker activation by pathogen-associated molecular patterns (PAMPs) during BCG infection is responsible for the delay in CD8+ T cell priming. Using rOVA-expressing bacteria, our results indicate that infection of mice with BCG-OVA generates greatly reduced levels of OVA, which are 70-fold lower in comparison to the levels generated during infection of mice with Listeria monocytogenes-expressing OVA. Furthermore, increasing the dose of OVA, but not PAMP signaling during BCG-OVA infection resulted in rapid Ag presentation and consequent expansion of the CD8+ T cell response, indicating that the generation of reduced Ag levels, not lack of PAMP-associated inflammation, was responsible for delayed priming of CD8+ T cells. There was a strong correlation between the relative timing of Ag presentation and the increase in the level of OVA in vivo. Taken together, these results reveal that some slowly replicating pathogens, such as mycobacteria, may facilitate their chronicity by generating reduced Ag levels which causes a substantial delay in the development of acquired immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8+ T cell is stimulated by APCs that harbor intracellular pathogens (viruses or intracellular bacteria) (1, 2). Pathogen-derived proteins in the intracellular milieu are processed through the MHC class I-processing pathway and presented to CD8+ T cells. The hallmark of the immune system is the specificity and the speed at which this process occurs (3, 4, 5). During infection with viruses such as lymphocytic choriomeningitis virus or with intracellular bacteria such as Listeria monocytogenes (LM),3 Ag presentation is over within the first few days of infection, implying that the programming for the development of CD8+ T cells occurs early on during the infection (3, 4, 5). Subsequent to potent Ag presentation, primed T cells undergo a massive phase of clonal expansion (>10,000-fold) which peaks at day 7 of infection (6). Subsequently, CD8+ T cell response undergoes a rapid contraction phase and only a small fraction of the primed CD8+ T cells (~5–10%) persist in the lymphoid organs as a stable pool of memory CD8+ T cells (7). The onset and kinetics of the contraction of CD8+ T cell response has been previously shown to be independent of the dose of the pathogen, the amount of Ag displayed and the duration of infection, implying that the contraction of the CD8+ T cell response is programmed very early during infection (8, 9).

We have previously shown that this paradigm of rapid CD8+ T cell differentiation is not strictly followed during infection of mice with the intracellular bacterium, Mycobacterium bovis (bacillus Calmette-Guérin (BCG)) (10, 11). CD8+ T cells that differentiate during infection of mice with BCG undergo limited activation during the first 7 days of infection, the response peaks during the third week of infection, followed by a protracted and reduced contraction phase. The delay in generating a rapid primary CD8+ T cell response against BCG could be due to the induction of inappropriate inflammatory signals by BCG, or due to the reduced generation of antigenic load in vivo. In this report, we have evaluated the influence of pathogen dose on the expansion and contraction of CD8+ T cell response against BCG. Our results indicate that the infection of mice with a higher dose of BCG results in rapid priming of CD8+ T cell response which occurs ~1–2 wk earlier than with the low dose and is followed by increased contraction. In contrast, infection of mice with different doses of LM does not influence the onset of expansion and the onset and extent of contraction subsequently. Thus, our results indicate that for pathogens that display poor in vivo growth (such as BCG), the dose of the pathogen can enormously modulate the differentiation of CD8+ T cell response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacterial strains

BCG-OVA is a previously described recombinant strain (12) engineered with a partial sequence of the OVA gene (codons 230–359), downstream of the Ag 85B secretion signal (13), under the control of heat shock protein 60 promoter (14). Codons 230–359 of the OVA gene encode the SIINFEKL epitope (OVA257–264) and its flanking sequences (15). Single colonies were used to inoculate liquid cultures, which were incubated at 37°C under constant shaking in 7H9 medium containing glycerol (0.2%), Tween 80 (0.05%), and albumin-dextrose supplement (10%; Difco). At mid-log phase (OD600 = 1.0), bacteria were harvested and frozen at –80°C (in 20% glycerol). CFU were determined by plating serial dilutions in PBST (0.025% Tween 80) on Middlebrook 7H10 solid medium containing glycerol (0.5%) and oleic acid-albumin-dextrose supplement (10%; Difco).

OVA-expressing LM (LM-OVA), as described previously (12), was grown to OD600 nm = 0.4. The bacteria were grown in brain heart infusion (BHI) medium (Difco), supplemented with 50 µg/ml streptomycin (Sigma-Aldrich). At mid-log phase (OD600 = 1.0), bacteria were harvested and frozen in 20% glycerol and stored at –80°C. CFU were determined by performing serial dilutions in 0.9% NaCl, which were spread on BHI-streptomycin agar plates.

Mice and immunizations

Female C57BL/6, 6–8 wk of age, were obtained from The Jackson Laboratory. B6.SJL (CD45.2–) and OT-1-transgenic mice (CD45.2+Thy1.2+ expressing the TCR of OVA257–264) were obtained from The Jackson Laboratory. The following mice were obtained from Taconic Farms through the National Institute of Allergy and Infectious Diseases Exchange Program, National Institutes of Health: OT-1/Rag-1, line no. 4175 (16, 17). Mice were maintained in the Animal Facility at the Institute for Biological Sciences (National Research Council of Canada) in accordance with the guidelines of the Canadian Council on Animal Care. For most experiments, mice were initially parked with 104 OT-1 CD8+ T cells and then challenged with LM-OVA or BCG-OVA. For immunizations with LM-OVA, frozen stocks were thawed and diluted in 0.9% NaCl and mice were inoculated with 1 x 104 LM-OVA suspended in 200 µl of 0.9% NaCl, via the lateral tail vein (i.v.). For BCG-OVA, frozen stocks were thawed and diluted in PBS containing 0.025% Tween 80 and injections were conducted through the i.v. route. Age-matched control mice were inoculated with 200 µl of PBS.

Assessment of bacterial burden in spleen

Single-cell suspensions from infected mice were tweezed in RPMI 1640 and an aliquot of the suspension was lysed with water for 30 s and then evaluated for the numbers of viable bacteria. For LM-OVA, CFU were determined by plating 100 µl of aliquots of serial 10-fold dilutions in 0.9% saline on BHI plates. For BCG-OVA, CFU was determined by plating 100 µl of aliquots of serial 10-fold dilutions in PBS containing 0.025% Tween 80 on 7H10 plates containing glycerol (0.5%) and oleic acid-albumin-dextrose supplement.

CD8+ T cell purification

Single-cell suspensions were prepared by tweezing the pooled spleens (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 (20 x 106/ml) in 0.5–1 ml of RPMI 1640 containing 8% FBS (HyClone Laboratories) and 50 µg/ml gentamicin (Invitrogen Life Technologies), hereafter referred to as R8-A. CELLection Biotin Binder Dynabeads precoated as per manufacturer’s instructions (Dynal Biotech), with biotin-conjugated rat anti-mouse CD8beta.2 mAb (53.5.8; BD Biosciences), were added to the resuspended cell pellet at a ratio of five beads to cell, and incubated for 15–20 min at 4°C in a rotating platform. CD8beta+ T cells were separated by magnetic isolation. Dynabead detachment was done using the CELLection Biotin Binder kit releasing buffer (DNase; 188 U/108 Dynabeads) in 37°C shaker for 15 min, followed by two to three rounds of washing/magnetic separation. This protocol resulted in >95% pure CD8+ T cells as determined by follow-up analysis with PE-conjugated rat anti-mouse CD8{alpha} (BD Biosciences). Analysis was performed using EPICS XL Flow Cytometer and EXPO software (Beckman Coulter).

Assessment of the fate and phenotype of Ag-specific CD8+ T cells

For evaluation of the phenotype of OVA257–264-specific CD8+ T cells in vivo, aliquots (10 x 106) of spleen cells were incubated in 200 µl of PBS plus 1% BSA with anti-CD16/32 at 4°C. After 10 min, cells were stained with various Abs (anti-CD8, anti-CD62L, anti-IL-2R{alpha}, anti-PD-1, anti-IL-7R{alpha}, anti-CD69, anti-mouse CD44, anti-CD45.2) for 30 min, on ice. All Abs were obtained from BD Biosciences. Cells were then washed with PBS-BSA and incubated for 30 min with PE-H-2KbOVA257–264 tetramer (Beckman Coulter) at room temperature. Cells were washed with PBS and fixed in 0.5% formaldehyde and acquired on FACSCanto using FACSDIVA software (BD Biosciences).

Assessment of cell cycling

Cell cycling was evaluated according to the protocol of Tough and Sprent (18) using the BD Biosciences BrdU staining kit. Three days before the harvesting of spleens from infected mice, BrdU was provided (0.08%) in the drinking water every day. Spleens were harvested, single-cell suspensions were prepared, and aliquots of spleen cells (10 x 106/ml) were stained with anti-CD8 Ab and H-2KbOVA257–264 tetramer for 30 min as described above. After staining, cells were washed, permeabilized, and incubated with DNase for 30 min at 37°C. Cells were then stained with anti-BrdU Ab on ice for 30 min, washed, fixed in 0.5% formaldehyde, and acquired on BD Biosciences FACS Canto analyzer.

Assessment of in vivo cytolytic activity

In vivo cytolytic activity of Ag-specific CD8+ T cells was enumerated according to recently developed methods (19, 20). Donor spleen-cell suspensions were prepared and RBC were lysed by ammonium chloride treatment. Cells were split into two aliquots and one of the aliquots was pulsed with OVA257–264 peptide (10 µg/ml) in R8 medium at 37°C for 30 min. Both populations were then stained with the dye PKH26 (4 µM) at room temperature for 3 min. The pulsed aliquot was stained with 10x CFSE (5 µM) and the unpulsed with 1x CFSE (0.5 µM) in PBS at room temperature in the dark for 8 min. The two aliquots were mixed 1:1 and injected (20 x 106/mouse) into recipient mice that were infected previously with LM-OVA or BCG-OVA. PBS-injected recipient mice served as controls. At 24 h after the donor cell transfer, spleens were removed from recipients and the relative numbers of peptide-pulsed vs control donor cells were enumerated according to a previously published equation (20).

Assessment of OVA expression in infected spleens by quantitative RT-PCR

Spleens were harvested from mice and snap-frozen in a dry ice/100% ethanol bath. Total RNA was extracted using the Qiagen RNeasy mini kit according to the manufacturer’s instructions along with rapid mechanical lysis. The whole spleen was cut into pieces and each piece was lysed in 1 ml of lysis buffer in a MiniBeadbeater 3110BX (BioSpec Products) with glass beads ({phi} = 0.5 mm and {phi} = 0.1 mm; BioSpec Products). Total RNA from homogenates was extracted according to the manufacturer’s instructions. RNA was treated with RNase-free DNase I (Roche Applied Science) for 30 min at 37°C. The DNase was removed using the Qiagen RNeasy mini kit according to the manufacturer’s instructions. Ten micrograms of total RNA was taken for cDNA synthesis. cDNA was synthesized using N8 random primers purchased from Sigma-Aldrich. RNA was made linear at 65°C for 5 min and cDNA was synthesized in a 40-µl reaction volume containing 3 µl of N8 random primers (5 µg/µl), 8 µl of 5x first strand buffer, 4 µl of DTT (100 mM), 5 µl of dNTP (10 mM), 1 µl of RNase OUT (40 U/µl), 2 µl of Superscript II (200 U/µl; Invitrogen Life Technologies), 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. Identical samples not treated with Superscript II were also prepared as controls to measure DNA contamination. Remaining 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 quantitative PCR SYBR Green supermix (ABgene). The primers used for the BCG 16S rRNA cDNA were 5'-CTGGGAAACTGGGTCGTAATAC-3' and 5'-CCGTCGTCGCCTTGGTAG-3' and for the truncated OVA cDNA within BCG-OVA, 5'-CAACCTCACATCTGTCTTAATGG-3' and 5'-GCCTCTGCTGACCCTACC-3'. The primers used for the LM 16S rRNA cDNA were 5'-GCGCAGGCGGTCTTTTAAG-3' and 5'-CAATGACCCTCCCCGGTTA-3' and for the OVA cDNA within LM-OVA 5'-CAGCCAAGCTCCGTGGATT-3' and 5'-TCTCCCACAGTCCTTTGAAGACA-3'. Primers were designed using Beacon Designer 4.0. All default conditions were used except the fragment length was changed to 75–150 bp. To obtain a standard curve for each primer-template set, 10-fold dilutions of known amounts of BCG-OVA or LM-OVA chromosomal DNA (0.01, 0.1, 1, 10, and 100 attomoles) were used as template DNA. This standard curve was run together with triplicate reactions of the uncharacterized samples. PCR conditions were optimized based on the dissociation curve of each primer and its target. PCR was performed in sealed tubes in a 96-well microtiter plate in an iCycler iQ Thermocycler (Bio-Rad). The 26-µl reaction consisted of 12.5 µl of quantitative PCR SYBR Green supermix (ABgene), 1.2 µl of each primer, 9.1 µl of DNase/RNase-free H2O, and 1 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 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. Fluorescence was measured during the annealing step and plotted against the amplification cycle. Absolute quantitative analysis of the data was extrapolated from the standard curve. Primer efficiencies were between 98 and 100%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Naive and memory CD8+ T cells respond to BCG-OVA infection with delayed kinetics

CD8+ T cells from naive OT-1 TCR-transgenic mice were parked in naive hosts which were challenged i.v. with 104 LM-OVA or BCG-OVA (Fig. 1A). The expansion in the numbers of OVA-tetramer+CD8+ T cells was evaluated kinetically at various time intervals. Although LM-OVA infection induced a rapid expansion in OVA-specific CD8+ T cell response, little expansion in the numbers of OVA-specific CD8+ T cells was noted within the first 7 days in mice that were infected with 104 BCG-OVA (Fig. 1A). The response against BCG-OVA peaked between days 14 and 21 and displayed little signs of contraction subsequently. Considering that memory CD8+ T cells are more sensitive than naive CD8+ T cells in their responsiveness to antigenic encounter, we therefore determined whether memory CD8+ T cells would respond rapidly to BCG-OVA. Memory CD8+ T cells were generated by infecting OT-1-parked C57BL/6J mice with LM-OVA as described above. At day 60, CD8+ T cells were purified (~10% OVA-tetramer+ cells) and these cells were adoptively transferred into naive B6.SJL mice (CD45.2–) that were challenged i.v. with 104 LM-OVA or BCG-OVA. These OVA-specific memory CD8+ T cells proliferated rapidly in mice that were challenged with LM-OVA (Fig. 1B). However, as with the response of naive CD8+ T cells (Fig. 1A), memory CD8+ T cells (Fig. 1B) responded to BCG-OVA infection with delayed kinetics which peaked during the second to third week of infection.


Figure 1
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  		FIGURE 1. Naive and memory CD8 T cells respond to BCG-OVA infection with delayed kinetics. Naive C57BL/6J mice were injected with 1 x 104 CD8+ cells from naive OT-1 TCR-transgenic mice, infected the next day with 104 LM-OVA or BCG-OVA, and the response of naive CD8+ T cells was evaluated (A). OVA257–264-specific memory CD8+ T cells were generated in C57BL/6J mice after injection with OT-1 cells and infection with LM-OVA. At day 60 of infection, CD8+ T cells were purified from these memory mice (~10% OVA-specific) and transferred (~5 x 105 OVA-specific CD8+ T cells/mouse) into naive B6.SJL (CD45.2–) mice that were challenged (104, i.v.) with LM-OVA or BCG-OVA. At various time intervals, the percentages of donor (CD45.2+) tetramer+CD8+ T cells were enumerated (B). At various time intervals, spleens were removed from the recipient mice and the numbers of OVA257–264-specific CD8+ T cells were evaluated after staining with H-2KbOVA257–264 tetramers and anti-CD8 and anti-CD44 Abs. Each time point involved evaluation of three mice per group.

 
Rapid response of CD8+ T cells during infection with 106 dose of BCG-OVA

C57BL/6 mice were parked with OT-1 cells and challenged with LM-OVA or BCG-OVA. For LM-OVA, 102 and 104 doses were used. Doses of LM-OVA that are higher than 104 are lethal for mice. For BCG-OVA, 104 and 106 doses were used. Infection with an even lower dose of BCG-OVA, 102, does not result in any detectable response in this model (our unpublished observations). During infection with LM-OVA, hosts that were infected with a higher dose mounted a CD8+ T cell response that was greater in magnitude, but, the response peaked at day 7 irrespective of the dose used. Furthermore, the kinetics and extent of contraction of the CD8+ T cell response was similar for mice infected with a high or low dose of LM-OVA (Fig. 2, A and B). In contrast, the dose of BCG-OVA used made an enormous difference in the timing and the extent of expansion and subsequent contraction of CD8+ T cell response. Unlike the 104 dose of BCG-OVA where the CD8+ T cell response peaked around the second to third week, infection with the 106 dose of BCG-OVA resulted in a rapid CD8+ T cell response which peaked at day 7 and was followed by a rapid phase of contraction subsequently (Fig. 2, A and B). Thus, infection of mice with the 106 dose of BCG-OVA resulted in rapid kinetics of CD8+ T cell response that was similar to the one induced against LM-OVA. However, even with the 106 dose of BCG-OVA, the CD8+ T cell response was not as potent in magnitude as that induced against LM-OVA. As expected, higher dose of the pathogen resulted in a higher pathogen burden in the spleen (Fig. 2C).


Figure 2
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  		FIGURE 2. Differential effects of the dose of LM-OVA vs BCG-OVA on CD8+ T cell response. C57BL/6J mice were injected with OT-1 CD8+ T cells and infected subsequently with different doses of LM-OVA or BCG-OVA. At various time intervals, spleens were harvested and stained with anti-CD8 and OVA tetramers. Numbers in the panel (A) indicate the percentage of OVA tetramer-positive cells within CD8+ T cells at day 7 after infection. The relative percentages of OVA tetramer+CD8+ T cells in the various groups of mice at various time intervals was evaluated (B). Bacterial burden was evaluated at various time intervals in the spleens of mice infected with different doses of LM-OVA and BCG-OVA (C).

 
A 106 dose of BCG-OVA induces increased activation of CD8+ T cells

We evaluated whether the change in the kinetics of CD8+ T cell response noted during infection with the higher dose of BCG-OVA could be due to increased activation of primed CD8+ T cells. Expression of activation markers, IL-2R{alpha} (Fig. 3A), CD69 (Fig. 3B), and PD-1 (Fig. 3C) was evaluated at various time intervals during the activation of OVA-specific CD8+ T cells. As expected, CD8+ T cells differentiating during LM-OVA infection underwent a rapid phase of activation that was characterized by overwhelming up-regulation of IL-2R{alpha}, CD69, and PD-1. Interestingly, at day 7 of LM-OVA infection, the majority of the OVA-specific CD8+ T cells did not express IL-2R{alpha}, indicating rapid, but transient, activation of CD8+ T cells. OVA-specific CD8+ T cells induced during infection of mice with the 104 dose of BCG-OVA displayed little up-regulation of IL-2R{alpha}, CD69, or PD-1. In contrast, infection of mice with a 106 dose of BCG-OVA resulted in a detectable up-regulation of CD69 and PD-1, but not IL-2R{alpha}. Thus, upon comparing the expression of various activation molecules, it appears that CD8+ T cells undergo increased activation in mice infected with the 106 dose of BCG-OVA.


Figure 3
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  		FIGURE 3. Influence of BCG-OVA dose on activation of CD8+ T cells. C57BL/6J recipient mice were injected with OT-1 CD8+ T cells and subsequently infected with LM-OVA or BCG-OVA. At various time intervals, spleens were harvested from the recipient mice and the spleen cells were stained with anti-CD8 Ab and OVA tetramers and Abs against CD25 (A), CD69 (B), and PD-1 (C). Each time point involved the evaluation of four mice per group. The graphs show the average percentage of the cell surface expression of the given marker on the OVA-specific CD8+ T cells of the four mice at each given time point.

 
We also evaluated the expression of CD44, CD62L, and IL-7R{alpha} on CD8+ T cells because the changes in the expression of these molecules is linked to the differentiation of CD8+ T cells (21, 22, 23). To evaluate the phenotype of OVA-specific CD8+ T cells that are induced during BCG-OVA infection, we performed multicolor analysis by staining spleen cells simultaneously with PE-OVA tetramers and Abs against CD8, CD44, CD62L, IL-7R{alpha}. CD8+ T cells that were OVA-tetramer+ and CD44high were gated and the relative expression of CD62L vs IL-7R{alpha} was evaluated on gated cells. In response to LM-OVA infection, OVA-specific CD8+ T cells rapidly down-regulated the expression of CD62L as well as IL-7R{alpha} (Fig. 4). In the long-term, the majority of OVA-specific CD8+ T cells displayed the central memory phenotype (CD62LhighIL-7R{alpha}high). In contrast, at day 7 after BCG-OVA infection, the majority of OVA-specific CD8+ T cells did not down-regulate CD62L, and only a small proportion of cells down-regulated IL-7R{alpha}. The cells were not naive in phenotype because the expression was evaluated on gated CD44high cells. At subsequent time intervals, increased numbers of OVA-specific CD8+ T cells displayed reduced IL-7R{alpha} expression, while the cells continued to express high levels of CD62L.


Figure 4
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  		FIGURE 4. Unique differentiation of OVA-specific CD8+ T cells during infection with BCG-OVA. C57BL/6J recipient mice were injected with OT-1 CD8+ T cells and subsequently infected with LM-OVA or BCG-OVA. At various time intervals, spleens were harvested from the recipient mice and the spleen cells were stained with anti-CD8 Ab and OVA tetramers, and Abs against CD44, CD62L, and IL-7R{alpha}. Expression of CD62L vs IL-7R{alpha} was evaluated on cells that were CD8+ OVA tetramer+CD44high. Each time point involved the evaluation of four mice per group.

 
We also determined the expression of CD62L and CD44 on naive OT-1 CD8+ T cells after transfer into uninfected mice. Because naive OT-1 CD8+ T cells do not proliferate in the absence of Ag, increased numbers of naive OT-1 CD8+ T cells were injected (106/mouse) into uninfected mice and their expression was evaluated 7 days later. As is shown in Fig. 5A, naive OT-1 CD8+ T cells expressed low levels of CD44 and high levels of CD62L. They also expressed high levels of IL-7R{alpha} (data not shown). In response to infection, the expression of CD62L and IL-7R{alpha} was rapidly down-regulated on CD8+ T cells that were stimulated during infection with a 104 dose of LM-OVA (Fig. 5, B and C). In the longer term, a significant proportion of OVA-specific CD8+ T cells expressed high levels of CD62L and IL-7R{alpha}. Infection of mice with the 104 dose of BCG-OVA resulted in moderate up-regulation of CD44 (indicative of delayed differentiation) and little down-regulation of CD62L and IL-7R{alpha} at day 7. At subsequent time intervals, CD44 expression was up-regulated further on OVA-specific CD8+ T cells but the cells did not down-regulate CD62L enormously, indicating that muted activation of CD8+ T cells results in increased differentiation of CD8+ T cells into the central phenotype (11). The down-regulation of IL-7R{alpha} on OVA-specific CD8+ T cells was noted only after day 14, indicative of protracted differentiation. Interestingly, OVA-specific CD8+ T cells continued to express reduced levels of IL-7R{alpha} even in the longer term. Increasing the dose of BCG-OVA to 106 resulted in increased up-regulation of CD44 and down-regulation of CD62L (Fig. 5B). More importantly, the down-regulation of IL-7R{alpha} expression was noticeable even at day 7 after infection, similar to LM-OVA infection (Fig. 5C), indicating rapid differentiation of CD8+ T cells with the higher dose of BCG-OVA. Taken together, these results indicate that CD8+ T cells that differentiate during infection with the 106 dose of BCG-OVA receive a greater level of stimulation.


Figure 5
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  		FIGURE 5. Rapid differentiation of CD8+ T cells after infection with a higher dose of BCG-OVA. C57BL/6J recipient mice were injected with 1 x 106 OT-1 CD8+ T cells (A) or 1 x 104 OT-1 CD8+ T cells (B and C). Mice were left untreated (A) or infected with LM-OVA or BCG-OVA (B and C). At various time intervals, spleens were harvested from the recipient mice and the spleen cells were stained with anti-CD8 Ab and OVA tetramers, and Abs against CD44 and CD62L (A and B) and IL-7R{alpha} (C). Each time point involved the evaluation of four mice per group. The graphs show the average percentage of the cell surface expression of the given marker on the OVA-specific CD8+ T cells of the four mice at each given time point.

 
A 106 dose of BCG-OVA induces rapid cycling of CD8+ T cells

We speculated that if a higher dose of BCG-OVA results in increased activation of primed CD8+ T cells, then the primed cells may be undergoing a greater degree of proliferation. We determined the relative proliferation of OVA-specific CD8+ T cells induced against LM-OVA vs BCG-OVA by measuring the incorporation of BrdU which was provided in the drinking water 3 days previously. OVA-specific CD8+ T cells induced against LM-OVA displayed massive proliferation at day 7 after infection with nearly all OVA-specific CD8+ T cells incorporating BrdU (Fig. 6). This massive Ag-induced burst of proliferation was over rapidly and was followed by a low level (~5–10%) homeostatic proliferation of OVA-specific CD8+ T cells. OVA-specific CD8+ T cells induced against a 104 dose of BCG-OVA displayed reduced, but prolonged, phase of cycling in the first 3 wk of infection. Infection with the 106 dose of BCG-OVA, in contrast, resulted in an increased burst of proliferation of OVA-specific CD8+ T cells that was evident even at day 7 after infection, a situation similar to the response induced in the LM-OVA infection model. After the fourth week of infection, the proliferation of OVA-specific CD8+ T cells was not sustained at high levels in mice infected with a 104 or 106 dose of BCG-OVA.


Figure 6
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  		FIGURE 6. Infection with the higher dose of BCG-OVA results in rapid cycling of OVA-specific CD8+ T cells. C57BL/6J mice were injected with OT-1 CD8+ T cells followed by infection with LM-OVA or BCG-OVA. Three days before the harvest of spleens, BrdU (0.8 mg/ml) was incorporated into the drinking water of mice which was changed daily. Spleen cells were stained with various Abs and OVA tetramers, followed by intracellular staining for BrdU. Numbers in the panels indicate the percentage of BrdU+CD8+ T cells among the tetramer+ cells (A). The relative changes in BrdU incorporation among OVA-tetramer+CD8+ T cells was evaluated at various time points after infection (B). Data are derived from analysis of individual spleens (n = 3) per time point.

 
Rapid development of functional CD8+ T cell response after infection with a 106 dose of BCG-OVA

It is possible that the CD8+ T cells may be induced rapidly during infection with the 106 dose of BCG-OVA, but they may not be functional. To determine this, we measured the cytolytic activity of CD8+ T cells in a physiological setting using the recently developed assay which measures killing of target cells in vivo (19, 20). These experiments were done without the transfer of OT-1-transgenic CD8+ T cells. On day 7 after LM-OVA infection, CD8+ T cells displayed high in vivo cytolytic activity toward specific targets resulting in complete elimination (~100%) of specific targets (Fig. 7). The extent of this cytolytic response was reduced at subsequent time intervals as only 30–40% of specific targets were eliminated. Infection of mice with a 104 dose of BCG-OVA resulted in little in vivo cytolytic activity (~10%) at day 7. This response increased progressively at subsequent time intervals, peaked at the third week of infection, and persisted subsequently at levels similar to those induced during LM-OVA infection. Infection with the 106 dose of BCG-OVA in contrast resulted in the development of rapid in vivo cytolytic response (~70%) at day 7. Thus, infection of mice with 106 dose of BCG-OVA results in rapid development of functional CD8+ T cells. We also measured the frequency of OVA-specific IFN-{gamma}-secreting CD8+ T cells at various time intervals after LM-OVA or BCG-OVA infection. Infection of mice with the lower dose of BCG-OVA resulted in a delayed IFN-{gamma}-secreting CD8+ T cell response that peaked during the second to third week of infection (Fig. 7C). In contrast, infection of mice with the higher dose of BCG-OVA resulted in a rapid induction of IFN-{gamma}-secreting OVA-specific CD8+ T cells. Thus, even without the transfer of OT-1 cells, naive CD8+ T cells respond differently to varying doses of BCG-OVA.


Figure 7
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  		FIGURE 7. Infection of mice with a higher dose of BCG-OVA results in a rapid development of functional CD8+ T cells. C57BL/6 mice were infected i.v. with LM-OVA or BCG-OVA in the absence of OT-1 cells. At various time intervals, peptide-pulsed, and PKH26 and CFSE-labeled spleen cells from naive donor mice were injected into naive mice, or into mice that were preinfected with LM-OVA or BCG-OVA. Spleens were removed from recipients at 24 h posttransfer and the relative numbers of peptide-pulsed vs control cells was enumerated in individual spleens. The relative profile of the donor populations in LM-OVA- or BCG-OVA-infected mice at day 7 is shown (A). The relative changes in the percent killing of specific donor cells at various time intervals of LM-OVA- and BCG-OVA-infected mice was enumerated (B). The frequency of IFN-{gamma}-secreting OVA-specific CD8+ T cells was enumerated by ELISPOT assay (C). Data are derived from analysis of individual spleens (n = 3) per time point. Experiment is representative of three such experiments conducted.

 
Additional inflammatory mediators do not accelerate the priming of the CD8+ T cell response against BCG-OVA

Infection with increased dose of the pathogen would result in an increase in two important components. First, there would be increased levels of bacterial lipids, pathogen-associated molecular patterns (PAMPs), which would result in increased engagement of receptors and consequent induction of inflammatory cytokines. Second, increased pathogen burden would result in increased levels of Ag. We evaluated whether the increased levels of PAMPS are responsible for rapid CD8+ T cell differentiation that was noted during infection with the 106 dose of BCG-OVA. Mice were infected with a 104 dose of BCG-OVA and injected with CpG, poly I:C, or LM. All of these immune stimulators were injected i.p. A single dose of LM was administered i.p. on the day of BCG-OVA infection. In the case of CpG and poly I:C, mice received one dose on the day of BCG-OVA infection and an additional dose 3 days later. Addition of these immune stimulators did not accelerate the development of CD8+ T cell response during infection with the 104 dose of BCG-OVA (Fig. 8). Injection of mice with CpG resulted in massive increases in the spleen cell numbers (Fig. 8C). The experiments described above were performed with immune stimulators that are different from those present in BCG. We therefore tested whether the PAMPs present in BCG itself may be the reason behind the rapid differentiation of CD8+ T cells that was noted with the increased dose of BCG-OVA. We therefore infected one group of mice with a mixture of 106 BCG and 104 BCG-OVA. Mice that were infected only with 104 BCG-OVA served as controls. Although coinjection of mice with the 106 dose of BCG and 104 BCG-OVA resulted in increased spleen cell numbers, this did not accelerate the development of the OVA-specific CD8+ T cell response (Fig. 8, D and E).


Figure 8
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  		FIGURE 8. Engagement of additional inflammatory mediators does not influence the kinetics of CD8+ T cell differentiation against BCG-OVA. C57BL/6J mice were injected with OT-1 CD8+ T cells followed by infection with BCG-OVA (104, i.v.). A–C, On days 0 and 3, some groups of mice received CpG i.p. (50 µg/mouse) or poly I:C (50 µg/mouse). Control mice received PBS. One group of mice received LM (104, i.p.) given on day 0 of infection. D and E, On day 0, some mice received BCG (106, i.v.) and control mice received PBS. The percentage of OVA-specific CD8+ T cells was determined by gating on CD8+ cells followed by further gating on tetramer+ cells. Each time point involved the evaluation of three mice per group per time point. Average percentages (A) and absolute numbers (B and D) of OVA-specific CD8+ T cells were enumerated for the various groups of mice. Number of cells in the spleens of infected mice was evaluated at various time intervals after infection (C and E).

 
Both bacterial burden and bacterial RNA levels decrease with time

We then sought to determine the amount of OVA that was present in the infected spleens at various time intervals after BCG-OVA infection. We noted that the ratio of total bacterial RNA (16S) to the bacterial burden (CFU) remains stable during the course of LM-OVA or BCG-OVA infection (data not shown). We also noted that the expression of OVA mRNA remains stable in comparison to the total bacterial RNA for both LM-OVA as well as BCG-OVA infections (data not shown). Because the bacterial burden declines progressively with time (Fig. 2C), the absolute amounts of total bacterial RNA (16S) and OVA mRNA also decline progressively (Fig. 9, A–C). During the BCG-OVA infection, RNA levels drop conspicuously after the first few weeks of infection and the levels remain stable thereafter. Infection with the 106 dose of BCG-OVA results in higher amounts of total bacterial (16S) RNA and OVA mRNA. We calculated the relative peak levels of OVA mRNA generated in mice infected with LM-OVA or BCG-OVA (Fig. 9D). Infection of mice with the 104 dose of BCG-OVA resulted in the generation of OVA mRNA that was ~70-fold less in comparison to that generated during infection of mice with 104 LM-OVA. Increasing the dose of BCG-OVA to 106 increased the level of OVA mRNA, but the levels were still less than those generated during LM-OVA infection. (Fig. 9D). The relative drop in the levels of OVA mRNA in the long-term was similar to the drop in the bacterial burden (CFU) (Fig. 9E).


Figure 9
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  		FIGURE 9. Increased OVA mRNA expression after infection of mice with higher dose of BCG-OVA. C57BL/6J mice were infected with LM-OVA (104), BCG-OVA (104), or BCG-OVA (106). At various time intervals, total bacterial RNA (16S rRNA) and OVA mRNA were quantified from spleens as explained in Materials and Methods. The relative increase in the expression of RNA in comparison to a naive mouse was determined at various time intervals for mice infected with 104 BCG-OVA (A), 106 BCG-OVA (B), or 104 LM-OVA (C). The relative expression of peak levels of OVA mRNA was determined for the three groups of mice (D). Fold reduction in the level of OVA mRNA and the bacterial burden (CFU) from days 7 to 60 was plotted for mice infected with 104 BCG-OVA or 106 BCG-OVA (E).

 
Infection with the 106 dose of BCG-OVA induces early Ag presentation

Considering that Ag presentation is the key event that drives the differentiation of CD8+ T cells, we wanted to determine the onset and duration of Ag presentation during infection of mice with the 104 and 106 dose of BCG-OVA. We have previously reported the in vivo model of Ag presentation wherein naive CFSE-labeled OT-1 TCR-transgenic CD8+ T cells are transferred into recipient mice that are previously infected with BCG-OVA. The proliferation of donor OT-1 cells is evaluated 4 days after transfer. Because proliferation of OT-1 cells is measured only 4 days after transfer, >20-fold greater numbers of OT-1 cells are transferred so that they can be detected even after transfer at day 60. Thus, these experiments measure Ag presentation to a large number of responding naive CD8+ T cells. Using this model, our results indicate that infection of mice with the 104 dose of BCG-OVA results in minimal proliferation of donor OT-1 cells within the first week of infection, which is followed by increased Ag presentation during the second week of infection, followed by gradual curtailment of Ag presentation subsequently (Fig. 10, A and B). In contrast, the onset, duration, and curtailment of Ag presentation occurred much earlier in the case of mice that were infected with the 106 dose of BCG-OVA (Fig. 10, A and B). When we evaluated the number of transferred OT-1 cells that had undergone more than seven divisions, the difference in the intensity of Ag presentation in low- and high-dose infected mice was even more appreciable (Fig. 10B). When we plotted OVA mRNA expression and Ag presentation (down-regulation of CFSE-labeled OT-1 cells) in the same graph, a good correlation was noted between OVA mRNA expression and Ag presentation (Fig. 10, C and D).


Figure 10
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  		FIGURE 10. Influence of BCG-OVA dose on the kinetics of Ag presentation. C57BL/6J mice were injected with BCG-OVA (104, i.v. or 106, i.v). At various time points post BCG-OVA infection, CFSE-labeled cells from an OT-1Rag1-transgenic donor mouse were injected into the recipient mice i.v. (8 x 106/mouse). After 4 days of transfer of the CFSE-labeled OT-1 cells, spleens were removed from the recipient mice and spleen cells stained with OVA tetramers and anti-mouse CD8{alpha} Ab. The expression of CFSE was assessed based on tetramer+ donor CD8+ T cells. Each time point involved the evaluation of four mice per group per time point. The percentage of CFSElow OVA-tetramer+ CD8+ T cells at day 4 of BCG-OVA infection is shown (A). The kinetic analysis of proliferation of the transferred OT-1 cells that underwent more than one or more than seven divisions (B) is shown. The relative level of OVA mRNA vs the proliferation of CFSE-labeled OT-1 cells that underwent >1 division was plotted in the case of mice that were infected with a 104 (C) or 106 (D) dose of BCG-OVA.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CD8+ T cells not only provide protection against viral infections (24), but they also facilitate protection against intracellular bacteria, such as LM, Mycobacterium tuberculosis, and Salmonella typhimurium (25, 26, 27). The hallmark of the immune system is the speed and the specificity with which the immune cells respond to counteract the rapid proliferation of pathogens. However, although the immune system is highly versatile in the sense that memory CD8+ T cells are induced against a wide variety of intracellular pathogens, we have previously shown that distinct differences in unique host-pathogen interactions can profoundly influence the speed and the differentiation of CD8+ T cells (10, 11, 28). In this report, we show that the rapidity of CD8+ T cell differentiation against an infection with BCG is influenced profoundly by the Ag dose. At an i.v. dose of 104, CD8+ T cell differentiation is minimal within the first 7 days of infection. Rapid CD8+ T cell differentiation occurs only at high doses and this is due to the increase in the amount of Ag, not inflammatory signals.

Increasing the antigenic dose is considered as the principal method of increasing the frequency of T cells and consequent vaccine efficacy. Using the various viral infection models, it has been shown that regardless of the dose of the pathogen used, CD8+ T cell responses always peak at day 7 after infection, and the extent of subsequent contraction of the response remains similar (1, 29). Although higher doses of pathogens induce quantitatively more numbers of memory CD8+ T cells, the slope of the CD8+ T cell differentiation curve (expansion and contraction) remains identical in case of infection with the low or high dose of the pathogen (1). Our results with the BCG model are unique as little differentiation of CD8+ T cells occurs within the first 7 days when mice are infected with the 104 dose and differentiation is accelerated when the dose is increased to 106. However, although the 104 dose of BCG given i.v. is relatively high upon comparison to other pathogens, BCG, unlike the majority of pathogens, doubles very slowly. The doubling time of BCG is >24 h (30) in comparison to other pathogens that double within minutes (~45 min for LM). Poor doubling of BCG would result in reduced generation of Ag within infected cells. Only after extensive proliferation of BCG-OVA in vivo would a sufficient amount of Ag be generated that would be adequate to induce a CD8+ T cell response. This is in agreement with a model proposed by Vijh et al. (31) wherein a critical antigenic threshold must be achieved to generate effective T cell priming.

Although both LM and BCG survive within APCs, they exhibit differences in their intracellular lifestyle (32). LM escapes from the phagosome and proliferates in the cytosol of infected cells (33). In contrast, BCG replicates inside the phagosome that poses a hostile and changing environment characterized by poor nutrient content, progressive decrease of the pH, and delivery of antibacterial peptides and lysosomal enzymes. The delay in CD8+ T cell priming during BCG-OVA infection may also be related to its phagosomal lifestyle. Indeed, it was previously reported that human monocytes chronically infected with BCG in vitro fail to present mycobacterial Ags, but not other Ags, to CD4+ T cells, suggesting that mycobacteria may actively sequester Ags from immune T cells that would allow replicating mycobacteria to persist in infected individuals (34). Thus, a slow replication rate coupled with the antigenic sequestration within phagosomes may result in minimal CD8+ T cell priming initially. However, our data indicate that infection with the 106 dose of BCG does induce rapid CD8+ T cell priming, implying that generation of sufficient antigenic level may be the most crucial requirement for rapid CD8+ T cell priming. However, although phagosomal residence of mycobacteria may facilitate antigenic sequestration, it has been previously reported that such phagosomes are leaky and consequently allow protein trafficking (35). It is therefore conceivable that infection with the higher dose of BCG-OVA results in a greater leakage of such phagosomes.

The influence of the level of Ag expression on the development of CD8+ T cell response has been evaluated previously in the context of a vaccinia virus infection model. In those studies, higher Ag expression by the vaccinia virus resulted in increased CD8+ T cell responses, and the responses were blunted if the antigenic levels were too high (36, 37). What we have shown here is that in the case of pathogens that replicate poorly, not only is the CD8+ T cell response increased in magnitude with the higher dose of BCG-OVA, but the response develops faster. An important point to be noted here is that despite the delay in CD8+ T cell priming with the 104 dose of BCG-OVA, a functional CD8+ T cell response does eventually develop by day 21, which persists at a magnitude that is similar to one induced against LM-OVA. So the uniqueness in the case of the CD8+ T cell response against the 104 dose of BCG-OVA manifests only in the kinetics and differentiation, rather than the magnitude and function of the response. This is based on our analysis of data in the absence of the transferred OT-1 cells. OVA-specific CD8+ T cells induced by BCG-OVA are highly cytolytic (Fig. 7), secrete IFN-{gamma} (11), and protect against a challenge with OVA-expressing tumor cells (12).

Injection of mice with CpG, poly I:C, LM, or BCG along with the 104 dose of BCG-OVA did not accelerate or amplify the CD8+ T cell response. This was surprising considering that such inducers of innate immune responses do influence T cell responses (38). The membrane of BCG is abundant in glycolipids that are highly stimulatory and are used as adjuvants (14, 39, 40, 41, 42). It is possible that injection of any additional PAMPs may not therefore result in further amplification of the response. Addition of such PAMPs may amplify T cell responses against vaccines that contain high Ag levels but lack in PAMPs. In the case of BCG-OVA infection, the situation is reversed as high levels of PAMPs are present under conditions of low Ag availability.

A surprising finding here is that even memory CD8+ T cells (generated after LM-OVA infection) are unable to proliferate rapidly when adoptively transferred into recipient mice and challenged with BCG-OVA infection. Thus, naive as well as memory CD8+ T cells display little response within the first week of BCG-OVA infection. Considering that memory CD8+ T cells display increased responsiveness to Ag, this indicates that the amount of Ag displayed during infection with the 104 dose of BCG-OVA must fall even below the levels necessary for engaging memory CD8+ T cells. This raises an important question: how can memory CD8+ T cells facilitate protection against such a pathogen?

Despite the prolonged BCG-OVA burden in vivo, the extent of Ag presentation decreased progressively. This correlated to the decrease in bacterial burden, bacterial RNA, and OVA expression after the second week of infection. Despite the reduction in the OVA mRNA, low-level expression of OVA mRNA was maintained. This supports our previous data that antigenic loss variants are not selected during BCG-OVA infection (10). Furthermore, the levels of IL-7R{alpha} on OVA-specific CD8+ T cells remained low during the chronic stages of BCG-OVA infection indicative of low-level Ag presentation. Chronic Ag presentation causes T cell exhaustion as has been reported in persistent viral infection models (43, 44). However, T cell exhaustion in viral models is strictly dependent on the persistence of high Ag levels (44). After the first few weeks of BCG-OVA infection, persistence of modest levels of OVA may not be sufficient to activate CD8+ T cells enormously in a chronic manner. Thus, CD8+ T cells may not be driven into exhaustion or deletion.

We have previously reported that CD8+ T cells that are induced against the 104 dose of BCG-OVA differentiate directly and mainly into the central phenotype CD62LhighCD44high (11). Results described in this study indicate that some of those cells display the central memory phenotype (CD62LhighCD44highIL-7R{alpha}high) and a significant proportion of cells also display an unusual phenotype (CD62LhighCD44highIL-7R{alpha}low). The differentiation of cells mainly into the central memory phenotype cells under such low Ag availability conditions makes sense because it results in the generation of the most useful memory subset (45), without massive contraction of the response (10, 11, 12). The immune system may need to preserve the maximal number of primed T cells when antigenic levels are limiting. The differentiation and role of CD8+ T cells that display the CD62LhighCD44highIL-7R{alpha}low phenotype is unclear. However, their numbers begin to decline only after 2 mo of continuous antibiotic therapy (our unpublished observations). These results indicate that despite the massive decline in bacterial and Ag burden, low-level Ag presentation may be occurring during BCG-OVA infection which results in the maintenance of cells expressing low levels of IL-7R{alpha}.

Our results highlight the complexities of unique host-pathogen interactions and their impact on CD8+ T cell differentiation. Mycobacteria may persist for prolonged periods in hosts due to their poor replication rate. This would result in the generation of low Ag levels, which may cause delayed and muted activation of T cells and therefore prevent complete elimination of the pathogen, favoring pathogen chronicity. From the host’s perspective, a delayed but significant T cell response is induced nonetheless. This response helps to control the pathogen levels substantially, although the pathogen is not eliminated completely (46). Due to the low Ag availability, T cells may not become functionally impaired. Such a host-pathogen interaction ensures survival for both the host and the pathogen and problems develop only when the host is immunosuppressed.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 a grant from the Canadian Institutes of Health Research. Back

2 Address correspondence and reprint requests to Dr. Subash Sad, Institute for Biological Sciences, National Research Council, 1200 Montreal Road, Building M-54, Ottawa, Ontario, Canada K1A 0R6. E-mail address: subash.sad{at}nrc.ca Back

3 Abbreviations used in this paper: LM, Listeria monocytogenes; BCG, bacillus Calmette-Guérin; BHI, brain heart infusion; PAMP, pathogen-associated molecular pattern. Back

Received for publication January 3, 2007. Accepted for publication April 20, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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